prof. aza gareth thomas. prof. aza 1. introduction 1. introduction the nucleic acids are the...
TRANSCRIPT
prof. azaprof. aza
Gareth ThomasGareth Thomas
prof. azaprof. aza
1. Introduction1. Introduction
•The nucleic acids are the The nucleic acids are the compounds that are compounds that are responsible responsible for the storage and transmission for the storage and transmission of the genetic information that of the genetic information that controls controls the growth, function and the growth, function and reproduction of all types of cellsreproduction of all types of cells..
•They are classified into two general They are classified into two general types: types: thethe deoxyribonucleic acids deoxyribonucleic acids (DNA), whose structures contain (DNA), whose structures contain the sugar residue the sugar residue β-β-D-deoxyribose; D-deoxyribose; and and the ribonucleic acids the ribonucleic acids (RNA), (RNA), whose structures contain the sugar whose structures contain the sugar residue residue β -β -D-ribose (Figure 10.1).D-ribose (Figure 10.1).
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prof. azaprof. aza
Figure 1. The structures of ββ -D-deoxyribose and ββ -D-ribose.
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nucleotide consists of a purine or nucleotide consists of a purine or pyrimidine basepyrimidine base
• Both types of nucleic acids are Both types of nucleic acids are polymers based on a repeating polymers based on a repeating structural unit known as a nucleotide structural unit known as a nucleotide (Figure 10.2). These nucleotides form (Figure 10.2). These nucleotides form long single-chain polymer molecules long single-chain polymer molecules in both DNA and RNA. in both DNA and RNA.
• Each Each nucleotide consists of a purine nucleotide consists of a purine or pyrimidine baseor pyrimidine base bonded to the 1’ bonded to the 1’ carbon atom of a sugar residue by a carbon atom of a sugar residue by a ββ -N-glycosidic link (Figure 10.3). -N-glycosidic link (Figure 10.3).
•These base-sugar subunits, These base-sugar subunits, which are which are known as nucleosidesknown as nucleosides, , are linked thare linked thrrough the 3’ and 5’ ough the 3’ and 5’ carbons of their sugar residues carbons of their sugar residues by phosphate units to form the by phosphate units to form the polymer chain.polymer chain.
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prof. azaprof. aza
Figure 2. The general structures of (a) nucleotides and (b) a schematic representation of a section of a nucleic acid chain.
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Figure 3. Examples of the structures of some of the nucleosides found in RNA. The ββ -N-glycosidic link is shaded The corresponding nucleosides in DNA are based on deoxyribose and use the same name but with the prefix deoxy.
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22.. Deoxyribonucleic Acids (DNA) Deoxyribonucleic Acids (DNA)
•DNA occurs in the nuclei of cells DNA occurs in the nuclei of cells in the form of a very compact in the form of a very compact DNA protein complex called DNA protein complex called chromatinchromatin. The protein in . The protein in chromatin consists mainly of chromatin consists mainly of histones, a family of relatively histones, a family of relatively small positively charged small positively charged proteins. proteins.
•The DNA is coiled twice around The DNA is coiled twice around as octomer of histone molecules as octomer of histone molecules with a ninth histone molecule with a ninth histone molecule attached to the exterior of these attached to the exterior of these minimini coils to form a structure like coils to form a structure like a row of heads spaced along a a row of heads spaced along a string (Figure 10.4). string (Figure 10.4).
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• This ‘string of beads’ is coiled and This ‘string of beads’ is coiled and twisted into compact structures known twisted into compact structures known as miniband units, which form the basis as miniband units, which form the basis of the structures of chromosomes. of the structures of chromosomes. Chromosomes are the structures that Chromosomes are the structures that form duplicates during cell division in form duplicates during cell division in order to transfer the genetic order to transfer the genetic information of the old cell to the two information of the old cell to the two new cells.new cells.
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prof. azaprof. aza
2.1 Structure2.1 StructureDNA molecules are large with relative molecular masses up to one trillion. The principal bases found in their structures are adenine (A), thymine (T), guanine (G) and cytosine (C), although derivatives of these bases are found in some DNA molecules (Figure 10.5). Those bases with an oxygen function have been shown to exist in their keto form.
• Figure 4. The ‘string of heads’ structure Figure 4. The ‘string of heads’ structure of chromatin. The DNA strand is of chromatin. The DNA strand is rround ound twice around each histone octomer. A twice around each histone octomer. A ninth histone molecule is bound to the ninth histone molecule is bound to the exterior surface of the coil.exterior surface of the coil.
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prof. azaprof. aza
DNA-binding DNA-binding proteinsproteins
Interaction of DNA with histones (shown in white, top). These proteins' basic amino acids (below left, blue) bind to the acidic phosphate groups on DNA (below right, red).
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Figure 5. The purine and pyrimidine bases found in DNA. The numbering is the same or each type of ring system.
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Structures of the four bases found in DNA and the nucleotide adenosine monophosphate.
AdenineGuanine
ThymineCytosine
Adenosine monophosphate
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•Chargaff showed that the molar Chargaff showed that the molar ratios of ratios of adenine to thymine and adenine to thymine and guanine to cytosine are always guanine to cytosine are always approximately 1: 1 approximately 1: 1 in any DNA in any DNA structure although the ratio of structure although the ratio of adenine to guanine varies adenine to guanine varies according to the species from according to the species from which the DNA is obtained. which the DNA is obtained.
• This and other experimental This and other experimental observations lead observations lead Crick and Watson Crick and Watson in in 1953 to propose that the three-1953 to propose that the three-dimensional structure of DNA consisted dimensional structure of DNA consisted of two single molecule polymer chains of two single molecule polymer chains held together in the form of held together in the form of a double a double helix by hydrogen bonding between helix by hydrogen bonding between the same pairs of bases,the same pairs of bases, namely: namely:
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•the the adenineadenine-- thymine thymine and and cytosine-guanine base pairs cytosine-guanine base pairs (Figure 10.6). These pairs of (Figure 10.6). These pairs of bases, which are referred to bases, which are referred to as as complementary base pairscomplementary base pairs, form , form the internal structure of the the internal structure of the helix. helix.
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prof. azaprof. aza
•They are hydrogen bonded in They are hydrogen bonded in such a manner that their flat such a manner that their flat structures lie parallel to one structures lie parallel to one another across the inside of the another across the inside of the helix. The two polymer chains helix. The two polymer chains forming the helix are aligned in forming the helix are aligned in opposite directions. In other opposite directions. In other wordswords,, at the ends of the at the ends of the structure one chain has structure one chain has a free 3’-a free 3’-OH group and the other chain OH group and the other chain has a free 5’-OH group. has a free 5’-OH group.
• X-ray diffraction studies have since X-ray diffraction studies have since confirmed that this is the basic three confirmed that this is the basic three dimensional shape of the polymer dimensional shape of the polymer chains of the β -DNA, the natural chains of the β -DNA, the natural form of DNA. form of DNA.
• This form of DNA has about This form of DNA has about ten ten bases per turn of the helix. bases per turn of the helix.
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• Its outer surface has two grooves Its outer surface has two grooves known as the minor and major known as the minor and major grooves, respectively, which act as grooves, respectively, which act as the binding sites for many ligands. the binding sites for many ligands. Two other forms of DNA, the A and Z Two other forms of DNA, the A and Z forms, have also been identified but forms, have also been identified but it is not certain if these forms occur it is not certain if these forms occur naturally in living cells.naturally in living cells.
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prof. azaprof. aza
• Electron microscopy has shown that Electron microscopy has shown that the double helical chain of DNA is the double helical chain of DNA is folded, twisted and coiled into quite folded, twisted and coiled into quite compact shapes. A number of DNA compact shapes. A number of DNA structures are cyclic and these structures are cyclic and these compounds are also coiled and twisted compounds are also coiled and twisted into specific shapes. These shapes are into specific shapes. These shapes are referred to as supercoils, supertwists referred to as supercoils, supertwists and superhelices as appropriate.and superhelices as appropriate.
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prof. azaprof. aza
The two strands of DNA are held together by hydrogen bonds between bases. The sugars in the backbone are shown in light blue.
• Figure 10.6. The double helical Figure 10.6. The double helical structure of B-DNA. Interchanging of structure of B-DNA. Interchanging of either the bases of a base pair and/or either the bases of a base pair and/or base pair with base pair does not base pair with base pair does not affect the geometry of this structure. affect the geometry of this structure. Reproduced from G. Thomas. Reproduced from G. Thomas. (Chemistry (Chemistry for Pharmacy and the Life Sciences including Pharmacology for Pharmacy and the Life Sciences including Pharmacology and Biomedical Science, I996, by per mission of Prentice and Biomedical Science, I996, by per mission of Prentice Hall, a Pearson Education Company.Hall, a Pearson Education Company.
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prof. azaprof. aza
33.. The General Functions of The General Functions of DNADNA
The DNA found in the nuclei of cells The DNA found in the nuclei of cells has three functions:has three functions:
(i) to act as a repository for the genetic (i) to act as a repository for the genetic information required by a cell to information required by a cell to reproduce that cell:reproduce that cell:
(ii) to reproduce itself in order to (ii) to reproduce itself in order to maintain the genetic pool when cells maintain the genetic pool when cells divide;divide;
(ii(iiii) to supply the information that the ) to supply the information that the cell requires to manufacture specific cell requires to manufacture specific proteins.proteins.
• Genetic information is stored in a form Genetic information is stored in a form known as genes known as genes by the DNA found in by the DNA found in the nucleus of a cell (see section 10.4).the nucleus of a cell (see section 10.4).
• The duplication of DNA is known as The duplication of DNA is known as replicationreplication. It results in the formation of . It results in the formation of two identical DNA two identical DNA mmolecuoleculles that carry es that carry the same genetic information from the the same genetic information from the original cell original cell
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• to the two new cells that are formed to the two new cells that are formed when a cell divides (see section when a cell divides (see section 10.5).10.5).
• The function of DNA in protein The function of DNA in protein synthesis is to act synthesis is to act as a template for as a template for the production of the various RNA the production of the various RNA molecules necessary to produce a molecules necessary to produce a specific protein (see section 6)specific protein (see section 6)
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prof. azaprof. aza
Figure 10.7. A schematic representation of the gene for the ββ -subunit of haemoglobin.
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4. Genes4. Genes
• Each species has its own internal and Each species has its own internal and external characteristics. These external characteristics. These characteristics are determined by the characteristics are determined by the information stored and supplied by information stored and supplied by the DNA in the nuclei of its cells. the DNA in the nuclei of its cells.
• This information is carried This information is carried in the in the form of a code based on the form of a code based on the consecutive sequences of bases consecutive sequences of bases found in sections of the DNA found in sections of the DNA structurestructure (see section 5). (see section 5).
• This code controls the production of This code controls the production of the peptides and proteins required the peptides and proteins required by the body. by the body.
• The The sequence of bases that act as sequence of bases that act as the code for the production of one the code for the production of one specific peptide specific peptide or protein molecule or protein molecule is known as is known as a gene.a gene.
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prof. azaprof. aza
Changing the sequence of the bases effect Changing the sequence of the bases effect on the external or internal characteristics of on the external or internal characteristics of an individualan individual
• Genes can normally contain from Genes can normally contain from several hundred to 2000 bases. several hundred to 2000 bases. Changing the sequence of the basesChanging the sequence of the bases in a gene by in a gene by adding, subtracting or adding, subtracting or changing one or more baseschanging one or more bases may may cause a change in the structure of cause a change in the structure of that protein with a subsequent that protein with a subsequent knock-on knock-on effect on the external or effect on the external or internal characteristics of an internal characteristics of an individualindividual. .
• For example, an individual may have For example, an individual may have brown instead of blue eyes or their brown instead of blue eyes or their insulin production may be inhibited, insulin production may be inhibited, which could result in that individual which could result in that individual suffering from diabetes. suffering from diabetes.
• A number of medical conditions have A number of medical conditions have been attributed been attributed to either the absence to either the absence of a gene or the presence of a of a gene or the presence of a degenerate or faulty genedegenerate or faulty gene in which in which one or more of the bases in the one or more of the bases in the sequence have been changed.sequence have been changed.
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prof. azaprof. aza
• In simple organisms, such as bacteria, In simple organisms, such as bacteria, genetic information is usually stored in genetic information is usually stored in a continous sequence of DNA bases. a continous sequence of DNA bases.
• However, in higher organisms the However, in higher organisms the bases forming a particular gene may bases forming a particular gene may occur in a number of separate occur in a number of separate sections sections known as exonsknown as exons, separated , separated by sections of DNA that do not appear by sections of DNA that do not appear to be a code for any process. to be a code for any process.
•These non-coding sections are These non-coding sections are referred to referred to as intronsas introns. For . For example, the gene responsible for example, the gene responsible for the β-subunit of haemoglobin the β-subunit of haemoglobin consists of 990 bases. These bases consists of 990 bases. These bases occur occur as three exons separated by as three exons separated by two introns two introns (Figure 10.7).(Figure 10.7).
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prof. azaprof. aza
•The complete set of genes that The complete set of genes that contain all the hereditary contain all the hereditary information of a particular information of a particular species is called a genome. species is called a genome.
•The Human Genome Project. The Human Genome Project. initiated in 1990initiated in 1990, sets out to , sets out to identify all the genes that occur identify all the genes that occur in human chromosomes and also in human chromosomes and also the sequence of bases in these the sequence of bases in these genes. genes.
•This will create an index that can This will create an index that can be used to locate the genes be used to locate the genes responsible for particular medical responsible for particular medical conditions. For example, the gene conditions. For example, the gene in region q31 of chromosome 7 is in region q31 of chromosome 7 is responsible for the protein that responsible for the protein that controls the flow of chloride ions controls the flow of chloride ions through the membranes through the membranes in the in the lungs. lungs.
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•The changing of about three The changing of about three bases in exon number 10 gives a bases in exon number 10 gives a degenerate gene that is known degenerate gene that is known to to bbe responsible for causing e responsible for causing cystic fibrosis cystic fibrosis in a large number in a large number of cases.of cases.
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prof. azaprof. aza
Figure 10.8. A schematic representation of the replication of DNA. The arrows show the direction of growth of the leading and lagging strands. Reproduced from G. Thomas, Chemistry for Pharmacy and the
Life Sciences including Pharmacology and Biomedical Science, 1996, by permission of Prentice Hall, a Pearson Education Company.
•Figure 10.8. A schematic Figure 10.8. A schematic representation of the replication of representation of the replication of DNA. The arrows show the direction DNA. The arrows show the direction of growth of the leading and of growth of the leading and lagging strands. lagging strands.
• Reproduced from G. Thomas, Chemistry for Pharmacy Reproduced from G. Thomas, Chemistry for Pharmacy and the Life Sciences including Pharmacology and and the Life Sciences including Pharmacology and Biomedical Science, 1996, by permission of Prentice Biomedical Science, 1996, by permission of Prentice Hall, a Pearson Education Company.Hall, a Pearson Education Company.
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DNA replication. The double helix (blue) is unwound by a helicase. Next, DNA polymerase III (green) produces the leading strand copy (red). A DNA polymerase I molecule (green) binds to the lagging strand. This enzyme makes discontinuous segments (called Okazaki fragments) before DNA ligase (violet) joins them together.
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5. Replication5. Replication
• Replication is believed to start with Replication is believed to start with the unwinding of a section of the the unwinding of a section of the double helix (Figure 10.8). double helix (Figure 10.8).
• Unwinding may start at the end or Unwinding may start at the end or more commonly in a central section more commonly in a central section of the DNA helix. of the DNA helix. It is initiated by the It is initiated by the binding of the DNA to specific binding of the DNA to specific receptor proteins receptor proteins that have been that have been activated by the appropriate first activated by the appropriate first messengemessenger r (see section 8.4). (see section 8.4).
• The separated strands of the DNA act The separated strands of the DNA act as as templates for the formation of a templates for the formation of a new daughter strandnew daughter strand. .
• Individual nucleotides, which are Individual nucleotides, which are synthesised in the cell by a complex synthesised in the cell by a complex route, bind by hydrogen bonding route, bind by hydrogen bonding between the bases to the between the bases to the complementary parent nucleotides. complementary parent nucleotides.
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•This hydrogen bonding is This hydrogen bonding is specific: specific: only the complementary only the complementary base pairs can hydrogen bond. base pairs can hydrogen bond.
• In other words, the hydrogen In other words, the hydrogen bonding can only bonding can only bbe between e between either either thymine and adenine thymine and adenine or or cytosine and guaninecytosine and guanine. .
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•This means that This means that the new the new daughter strand is an exact daughter strand is an exact replica of the original DNA strandreplica of the original DNA strand bound to the parent strand. bound to the parent strand.
•Consequently, replication will Consequently, replication will produce two identical DNA produce two identical DNA molecules.molecules.
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prof. azaprof. aza
• As the nucleotides hydrogen bond to As the nucleotides hydrogen bond to the parent strand they are linked to the parent strand they are linked to the adjacent nucleotide, which is the adjacent nucleotide, which is already hydrogen bonded to the already hydrogen bonded to the parent strand, by the action of parent strand, by the action of enzymes known as enzymes known as DNA polymerases. DNA polymerases.
• As the daughter strands grow, the As the daughter strands grow, the DNA helix continues to unwind. DNA helix continues to unwind.
• However, both daughter strands are However, both daughter strands are formed at the same time formed at the same time in the 5’ to in the 5’ to the 3’ direction. the 3’ direction.
• This means that the growth of the This means that the growth of the daughter strand that starts at the 3’ daughter strand that starts at the 3’ end of the parent strand can continue end of the parent strand can continue smoothly as the DNA helix continues smoothly as the DNA helix continues to unwind. to unwind.
• This strand is known the leading This strand is known the leading strand. However, this smooth growth strand. However, this smooth growth is riot possible for the daughter strand is riot possible for the daughter strand that started from the 5’ of the parent that started from the 5’ of the parent strand. strand.
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• This strand, known This strand, known as the lagging as the lagging strand,strand, is formed in a series of is formed in a series of sections, each of which still grows in sections, each of which still grows in the 5’ to 3’ direction. the 5’ to 3’ direction.
• These sections. which are known as These sections. which are known as Okazaki fragments after their Okazaki fragments after their discoverer, are joined together by discoverer, are joined together by the the enzyme DNA ligaseenzyme DNA ligase to form the to form the second daughter strand.second daughter strand.
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prof. azaprof. aza
• ReplicationReplication, , which starts at the end which starts at the end of a DNA helix, continues of a DNA helix, continues uuntil the ntil the entire structure has been duplicated. entire structure has been duplicated.
• The same result is obtained when The same result is obtained when replication starts at the centre of a replication starts at the centre of a DNA helix. DNA helix.
• In this case, In this case, unwinding continues in unwinding continues in both directions until the complete both directions until the complete molecule is duplicated.molecule is duplicated. This latter This latter situation is more common.situation is more common.
• DNA replication occurs when cell DNA replication occurs when cell division is division is imminentimminent. At the same time, . At the same time, new histones are synthesised. new histones are synthesised.
• This results in a thickening of the This results in a thickening of the chromatin filaments into chromosomes chromatin filaments into chromosomes (see section 2). These rod-like (see section 2). These rod-like structures can be stained and are large structures can be stained and are large enough to be seen under a microscope.enough to be seen under a microscope.
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prof. azaprof. aza
6. Ribonucleic Acids (RNA)6. Ribonucleic Acids (RNA)
• Ribonucleic acids are found in both Ribonucleic acids are found in both the the nucleus and the cytoplasmnucleus and the cytoplasm. In . In the cytoplasm RNA is located mainly the cytoplasm RNA is located mainly in small spherical organelles known in small spherical organelles known as as ribosome.ribosome. These consist of about These consist of about 65% RNA and 35% protein. 65% RNA and 35% protein.
• Ribonucleic acids are classified Ribonucleic acids are classified according to their general role in according to their general role in protein synthesis as: messenger RNA protein synthesis as: messenger RNA (mRNA): transfer RNA (tRNA): and (mRNA): transfer RNA (tRNA): and ribosomal RNA (rRNA). ribosomal RNA (rRNA).
• Messenger Messenger RNA informs the ribosome as RNA informs the ribosome as to what amino acids are required to what amino acids are required and and their order in the protein, that is, they their order in the protein, that is, they carry the genetic information necessary carry the genetic information necessary to produce a specific protein. to produce a specific protein.
• This type of RNA is synthesised as This type of RNA is synthesised as required and once its message has been required and once its message has been delivered it is decomposed. delivered it is decomposed.
prof. azaprof. aza
prof. azaprof. aza
Figure 10.9. (a) The general structure ol a section of an RNA polymer chain. (b) The hydrogen bonding between uracil and adenine. Reproduced from G.’Thomas, Chemistry to Pharmacy and the Life Science including Pharmacology including Biomedical Science, 1996, by permission of Prentice Hall, a Pearson Education Company.
• Figure 10.9. (a) The general structure Figure 10.9. (a) The general structure ooff a section of an RNA polymer chain. a section of an RNA polymer chain. (b) The hydrogen bonding between (b) The hydrogen bonding between uracil and adenine. uracil and adenine. Reproduced from Reproduced from G.’Thomas, Chemistry to Pharmacy and the Life Science G.’Thomas, Chemistry to Pharmacy and the Life Science including Pharmacology including Biomedical Science, including Pharmacology including Biomedical Science, 1996, by permission of Prentice Hall, a Pearson Education 1996, by permission of Prentice Hall, a Pearson Education Company. Company.
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prof. azaprof. aza
Figure 10.10. A schematic representation of a transcription process. Reproduced from G.’Thomas, Chemistry to Pharmacy and the Life Science including Pharmacology including Biomedical Science, 1996, by permission of Prentice Hall, a Pearson Education Company.
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• The structures of RNA molecules The structures of RNA molecules consist of a single polymer chain of consist of a single polymer chain of nucleotides with the same bases as nucleotides with the same bases as DNA, with the DNA, with the exception of thymine, exception of thymine, which is replaced by uracilwhich is replaced by uracil ( Figure ( Figure 9).9).
• These chains often contain single-These chains often contain single-stranded loops separated by short stranded loops separated by short sections of a distorted double helix sections of a distorted double helix (Figure 11). These structures are (Figure 11). These structures are known as hairpin loops. known as hairpin loops.
• All types of RNA are formed from DNA All types of RNA are formed from DNA by a process known by a process known as transcriptionas transcription. It . It is thought that the DNA unwinds and is thought that the DNA unwinds and the RNA molecule is formed in the 5’ to the RNA molecule is formed in the 5’ to 3’ direction. 3’ direction.
• It proceeds smoothly. with the 3’ end of It proceeds smoothly. with the 3’ end of the new strand bonding to the 5’ end of the new strand bonding to the 5’ end of the next nucleotide (Figure 10.10). the next nucleotide (Figure 10.10).
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prof. azaprof. aza
• This bonding is catalysed by enzymes This bonding is catalysed by enzymes known as known as RNA polymerasesRNA polymerases. .
• The sequence of bases in the new RNA The sequence of bases in the new RNA strand is controlled by the sequence of strand is controlled by the sequence of bases in the parent DNA strand. bases in the parent DNA strand.
• In this way In this way DNA controls the genetic DNA controls the genetic information being transcribed into the information being transcribed into the RNA molecule. RNA molecule.
• The strands of DNA also contain start The strands of DNA also contain start and stop signals, which control the size and stop signals, which control the size of the RNA molecule produced. of the RNA molecule produced.
• These signals are in the form of specific These signals are in the form of specific sequences of bases. sequences of bases.
• It is believed that the enzyme It is believed that the enzyme rhorho factor factor could be involved in the termination of could be involved in the termination of the synthesis and the release of some the synthesis and the release of some RNA molecules from the parent DNA RNA molecules from the parent DNA strand. However, in many cases there is strand. However, in many cases there is no evidence that this enzyme is involved no evidence that this enzyme is involved in the release of the RNA molecule.in the release of the RNA molecule.
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•The RNA produced within the The RNA produced within the nucleus by transcription is known nucleus by transcription is known as heterogeneousas heterogeneous nuclear RNA nuclear RNA (hnRNA), premessenger RNA (hnRNA), premessenger RNA (pre-mRNA) or primary transcript (pre-mRNA) or primary transcript RNARNA ( ptRNA). ( ptRNA).
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• Since the DNA gene from which it is Since the DNA gene from which it is produced contains both exons and produced contains both exons and introns, the hnRNA will also contain introns, the hnRNA will also contain its genetic information in the form of its genetic information in the form of a series of exons and introns a series of exons and introns complementary to those of its parent complementary to those of its parent gene.gene.
prof. azaprof. aza
prof. azaprof. aza
7. Messenger RNA (mRNA)7. Messenger RNA (mRNA)
• mmRNA RNA carries the genetic message from carries the genetic message from the DNA in the nucleus to a ribosomethe DNA in the nucleus to a ribosome. . This message instructs the ribosome to This message instructs the ribosome to synthesise a specific protein. synthesise a specific protein.
• mRNA is believed to be produced in the mRNA is believed to be produced in the nucnuclleus from hnRNA eus from hnRNA by removal of the by removal of the introns and the splicing together ointrons and the splicing together off the the remaining exons into a continuous remaining exons into a continuous genetic message, genetic message,
• the process being catalysed by the process being catalysed by specialised enzymes. The net result specialised enzymes. The net result is a smaller mRNA molecule with a is a smaller mRNA molecule with a continuous sequence of bases continuous sequence of bases that that are complementary to the gene’s are complementary to the gene’s exonsexons, this mRNA now leaves the , this mRNA now leaves the nucleus and carries its message in nucleus and carries its message in the form of a code to a ribosome.the form of a code to a ribosome.
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prof. azaprof. aza
prof. azaprof. aza
all protein synthesis starts with all protein synthesis starts with methioninemethionine• The code carried by mRNA was The code carried by mRNA was
broken in the 1960s by Nirenberg broken in the 1960s by Nirenberg and other workers. These workers and other workers. These workers demonstrated that demonstrated that each naturally each naturally occurring amino acid had a DNA code occurring amino acid had a DNA code that consisted of a sequence of three that consisted of a sequence of three consecutive bases known as a codonconsecutive bases known as a codon and that an amino acid could have and that an amino acid could have several different codons (Table 10.1), several different codons (Table 10.1),
• In addition, three of the codons are In addition, three of the codons are stop signals which instruct the stop signals which instruct the ribosome to sribosome to sttop protein synthesis. op protein synthesis.
• Furthermore, the codon Furthermore, the codon that initiates that initiates the synthesis is always AUG, which is the synthesis is always AUG, which is also the codon for methioninealso the codon for methionine. . Consequently, all protein synthesis Consequently, all protein synthesis starts with methionine. starts with methionine. However, few completed proteins However, few completed proteins have a terminal methionine because have a terminal methionine because this residue is normally removedthis residue is normally removed before the peptide chain is complete. before the peptide chain is complete.
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•Moreover, methionine can still Moreover, methionine can still bbe e incorporated in a peptide chain incorporated in a peptide chain because there are two different because there are two different tRNAs that transfer methionine tRNAs that transfer methionine to the ribosome (see section 8). to the ribosome (see section 8).
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prof. azaprof. aza
all living matter using the same genetic all living matter using the same genetic code for protein synthesiscode for protein synthesis
•One is specific for the transfer of One is specific for the transfer of the initial methionine whereas the the initial methionine whereas the other will only deliver methionine other will only deliver methionine to the developing peptide chain, to the developing peptide chain, By convention, the three letters By convention, the three letters of codon triplets are normally of codon triplets are normally written with their 5’ ends on the written with their 5’ ends on the left and their 3’ ends on the rightleft and their 3’ ends on the right..
•The mRNA’s codon code is known The mRNA’s codon code is known as the genetic code, Its use is as the genetic code, Its use is universal, universal, all living matter using all living matter using the same genetic code for protein the same genetic code for protein synthesis. synthesis.
•This suggests that all living matter This suggests that all living matter must have originated from the must have originated from the same source and is strong same source and is strong evidence for Darwin’s theory of evidence for Darwin’s theory of evolution.evolution.
prof. azaprof. aza
• Figure .11. The general structures of Figure .11. The general structures of tRNA. (a) The two-dimensional tRNA. (a) The two-dimensional cloverleaf representation showing some cloverleaf representation showing some of the invariable nucleotides that occur of the invariable nucleotides that occur in the same positions in most tRNA in the same positions in most tRNA molecules and (molecules and (bb) the three- ) the three- dimensional L shape dimensional L shape (From CHEMISTRY, by (From CHEMISTRY, by Linus Pauling and Peter Pauling. Copyright © 1975 by Linus Pauling and Peter Pauling. Copyright © 1975 by Linus Pauling and Peter Paling. Used with permission Linus Pauling and Peter Paling. Used with permission of W. H. Freeman and Company)of W. H. Freeman and Company)
prof. azaprof. aza
prof. azaprof. aza
8. Transfer RNA (tRNA)8. Transfer RNA (tRNA)
• tRNAs are also believed to be formed tRNAs are also believed to be formed in the nucleus from the hnRNA. in the nucleus from the hnRNA.
• They are relatively small molecules They are relatively small molecules that usually that usually contain from 73 to 94 contain from 73 to 94 nucleotides in a single strand. Some nucleotides in a single strand. Some of these nucleotides may contain of these nucleotides may contain derivatives of the principal bases, derivatives of the principal bases, such as such as 2’-O-methylguanosine (0MG) 2’-O-methylguanosine (0MG) and inosine (I).and inosine (I).
• The strand of tRNA is usually folded The strand of tRNA is usually folded into a into a three-dimensional L shapethree-dimensional L shape. .
•This structure, which consists of This structure, which consists of several loops, is several loops, is held in this shape held in this shape by hydrogen bondingby hydrogen bonding between between complementary base pairs in the complementary base pairs in the stem sections of these loops and stem sections of these loops and also by hydrogen bonding also by hydrogen bonding between bases in different loops. between bases in different loops.
prof. azaprof. aza
prof. azaprof. aza
Figure .11. The general structures of tRNA. (a) The two-dimensional cloverleaf representation showing some of the invariable nucleotides that occur in the same positions in most tRNA molecules and (b) the three- dimensional L shape (From CHEMISTRY, by Linus Pauling and Peter Pauling. Copyright © 1975 by Linus Pauling and Peter Paling. Used with permission of W. H. Freeman and Company)
• Figure .11. The general structures of Figure .11. The general structures of tRNA. (a) The two-dimensional tRNA. (a) The two-dimensional cloverleaf representation showing cloverleaf representation showing some of the invariable nucleotides some of the invariable nucleotides that occur in the same positions in that occur in the same positions in most tRNA molecules and (most tRNA molecules and (bb) the ) the three- dimensional L shape three- dimensional L shape (From (From CHEMISTRY, by Linus Pauling and Peter Pauling. CHEMISTRY, by Linus Pauling and Peter Pauling. Copyright © 1975 by Linus Pauling and Peter Copyright © 1975 by Linus Pauling and Peter Paling. Used with permission of W. H. Freeman and Paling. Used with permission of W. H. Freeman and Company)Company)
prof. azaprof. aza
•This results in the formation of This results in the formation of sections of double helical sections of double helical sstructures. tructures.
•However, the structures of most However, the structures of most tRNAs are represented in two tRNAs are represented in two dimensions dimensions as a cloverleaf as a cloverleaf (Figure (Figure 11).11).
prof. azaprof. aza
prof. azaprof. aza
•tRNA molecules tRNA molecules carry amino acid carry amino acid residues from the cell’s amino residues from the cell’s amino acid pool to the mRNA attached acid pool to the mRNA attached to the ribosome. to the ribosome.
•The amino acid residue is The amino acid residue is attached through an ester attached through an ester linkage to ribosome residue linkage to ribosome residue at at the 3’ terminal of the tRNA the 3’ terminal of the tRNA strandstrand, which almost invariably , which almost invariably has the sequence CCA. has the sequence CCA.
• This sequence plus a fourth nucleotide This sequence plus a fourth nucleotide projects beyond the double helix of the projects beyond the double helix of the stem. Each type of amino acid can only stem. Each type of amino acid can only be transported be transported byby its own specific tRNA its own specific tRNA molecule. molecule. In other words a tRNA that In other words a tRNA that carries serine residues will not carries serine residues will not transport alanine residuestransport alanine residues. In other . In other word, some amino acids can word, some amino acids can bbe carried e carried by several different tRNA moleculesby several different tRNA molecules
prof. azaprof. aza
prof. azaprof. aza
• The tRNA recognises the point on the The tRNA recognises the point on the mRNA where it has to deliver its mRNA where it has to deliver its amino acid through the use of a amino acid through the use of a group of three bases known group of three bases known as an as an anticodon. anticodon.
• This anticodon is a sequence of three This anticodon is a sequence of three bases found on one of the loops of bases found on one of the loops of the tRNA (Figure 11). the tRNA (Figure 11).
• The The anticodon can only form base anticodon can only form base with the complementary codon in the with the complementary codon in the mRNAmRNA. .
prof. azaprof. aza
•Consequently, the tRNA Consequently, the tRNA wwill only ill only hydrogen bond to the region of hydrogen bond to the region of the mRNA that has the correct the mRNA that has the correct codon, which means codon, which means its amino its amino acid can only be delivered to a acid can only be delivered to a specific point on the mRNA. specific point on the mRNA.
prof. azaprof. aza
• For example, a tRNA molecule with For example, a tRNA molecule with the the anantticodon CGA will only transport icodon CGA will only transport its alanine residue to a GCU codon its alanine residue to a GCU codon on on the mRNA.the mRNA.
• Furthermore, this mechanism will Furthermore, this mechanism will also control also control the order in which amino the order in which amino acid residues are added to the acid residues are added to the growing protein.growing protein.
prof. azaprof. aza
prof. azaprof. aza
99.. Ribosomal RNA (rRNA) Ribosomal RNA (rRNA)
• Ribosomes contain about 35% protein Ribosomes contain about 35% protein and 65% rRNA. and 65% rRNA.
• Their structures are complex and Their structures are complex and have not yet been fully elucidated. have not yet been fully elucidated.
• However, they have been found to However, they have been found to consist of two Sections that are consist of two Sections that are referred to referred to as the large and small as the large and small subunits. subunits.
• Each of these subunits contains Each of these subunits contains protein and rRNAprotein and rRNA
• In Eschericia coli the small subunit In Eschericia coli the small subunit has been shown to contain a 1542-has been shown to contain a 1542-nucleotide rRNA molecule whereas nucleotide rRNA molecule whereas the large contains two rRNA the large contains two rRNA molecules of 120 (Figure 12) and molecules of 120 (Figure 12) and 2094 nucleotides, respectively.2094 nucleotides, respectively.
prof. azaprof. aza
prof. azaprof. aza
• Experimental evidence suggests that Experimental evidence suggests that rRNA molecules have structures that rRNA molecules have structures that consist of consist of a single strand of a single strand of nucleotides whose sequence varies nucleotides whose sequence varies considerably from species to species. considerably from species to species.
• The strand is folded and twisted to The strand is folded and twisted to form a series of single-stranded loops form a series of single-stranded loops separated by sections of double helix separated by sections of double helix (Figure 12). (Figure 12).
prof. azaprof. aza
Figure 10.12. The proposed sequence of nucleotides in the 120-nucleotide subunit found in Escherichia coli ribosome showing the single-stranded loops and the double helical structures. (Reprinted, with permission, from the Annual Review of Biochemistry, volume 53 © I984 by Annual Reviews. www.Annual Reviews.org).
• The double helical segments are The double helical segments are believed to be formed by hydrogen believed to be formed by hydrogen bonding between complementary bonding between complementary base pairs. base pairs.
• The The general pattern of loops and general pattern of loops and helixes is very similar between helixes is very similar between species even though the sequence of species even though the sequence of nucleotides are differentnucleotides are different
prof. azaprof. aza
•However, little is known about However, little is known about the three-dimensional structures the three-dimensional structures of rRNA molecules of rRNA molecules and their and their interactions with the proteins interactions with the proteins found in the rifound in the ribbosome.osome.
prof. azaprof. aza
prof. azaprof. aza
10. Protein Synthesis10. Protein Synthesis
•Protein synthesis starts from the Protein synthesis starts from the N-terminal of the protein. N-terminal of the protein.
• IIt t proceeds in the 5’ to 3’ proceeds in the 5’ to 3’ direction along the mRNAdirection along the mRNA and and may be divided into four mayor may be divided into four mayor stages. namely: stages. namely: activation: activation: initiation: elongation: and initiation: elongation: and termination. termination.
ActivationActivation
•ActivationActivation is the formation of the is the formation of the ttRNA amino acid complex. RNA amino acid complex.
• InitiationInitiation is the binding of the is the binding of the mRNA to the ribosome and the mRNA to the ribosome and the activation of the ribosome. activation of the ribosome. ElongationElongation is the formation of the is the formation of the protein. protein.
prof. azaprof. aza
• TerminationTermination is the ending of the protein is the ending of the protein synthesis and its release from the synthesis and its release from the ribosome. ribosome.
• All these processes normally require All these processes normally require the participation of the participation of protein catalysts, protein catalysts, known as factors,known as factors, as well as other as well as other proteins whose function is not always proteins whose function is not always known. known.
• GTP and sometimes ATP act as sources GTP and sometimes ATP act as sources of energy of energy for the processes.for the processes.
prof. azaprof. aza
prof. azaprof. aza
10.1 Activation10.1 ActivationIt is believed that the amino acids from the cellular pool react with ATP to form an active amino acid-AMP complex. This complex reacts with the specific tRNA for the amino acid. the reaction being catalysed by a synthese that is specific for that amino acid.
• It is believed that the amino acidsIt is believed that the amino acids ( ( AAA) A) from the cellular pool react with from the cellular pool react with ATP to form ATP to form an active amino acid-AMP an active amino acid-AMP complexcomplex (AA-AMP) (AA-AMP). .
• This complex reacts with This complex reacts with the specific the specific tRNA for the amino acidtRNA for the amino acid, , the reaction the reaction being catalysed by being catalysed by a syntha synthaase se that is that is specific for that amino acid.specific for that amino acid.
prof. azaprof. aza
prof. azaprof. aza
Figure 13. A schematic representation of the initiation of protein synthesis.
prof. azaprof. aza
22.. Initiation Initiation• The general mechanism of initiation The general mechanism of initiation
is well documented but the liner is well documented but the liner details are still not known. details are still not known.
• It is thought that it starts with the It is thought that it starts with the two subunits of the ribosome two subunits of the ribosome separating and the binding of the separating and the binding of the mRNA to the smaller subunit. mRNA to the smaller subunit.
• Protein synthesis then starts by the Protein synthesis then starts by the attachment of a methionine-tRNA attachment of a methionine-tRNA complex to the mRNA complex to the mRNA so that it forms so that it forms the N-terminal of the new protein. the N-terminal of the new protein.
• Methionine is always the first amino Methionine is always the first amino acid in all protein synthesis because acid in all protein synthesis because its tRNA anticodon is also the signal its tRNA anticodon is also the signal for the ribosome system to start for the ribosome system to start protein synthesis. protein synthesis.
• Because Because the anticodon for the anticodon for methionine tRNA is UACmethionine tRNA is UAC, this , this synthesis will start synthesis will start at the AUG codon at the AUG codon of the mRNAof the mRNA. .
prof. azaprof. aza
•This codon is usually found This codon is usually found within the first 30 nucleotides of within the first 30 nucleotides of the mRNA. the mRNA.
•However, few proteins have an However, few proteins have an N-terminal methionine because N-terminal methionine because once protein synthesis has once protein synthesis has started the methionine started the methionine is usually is usually removed by hydrolysis. removed by hydrolysis.
prof. azaprof. aza
prof. azaprof. aza
• As soon as As soon as the methionine-tRNA has the methionine-tRNA has bound to the mRNAbound to the mRNA the larger the larger ribosomal subunit is believed to bind ribosomal subunit is believed to bind to the smaller subunit to the smaller subunit so that the so that the mRNA is sandwiched mRNA is sandwiched between the between the two subunits (Figure 13). two subunits (Figure 13).
• This large subunit is believed to have This large subunit is believed to have three binding sites called the three binding sites called the P P (peptidyl), A (acceptor) and E (exit) (peptidyl), A (acceptor) and E (exit) sites.sites.
• It attaches itself to the smaller It attaches itself to the smaller subunit so subunit so that its P site is aligned that its P site is aligned with the methionine- tRNA with the methionine- tRNA complex bound to the mRNAcomplex bound to the mRNA. .
•This P site is where the growing This P site is where the growing protein will be bound to the protein will be bound to the ribosomeribosome. .
prof. azaprof. aza
• The A site, which is thought to be The A site, which is thought to be adjacent to the P site, adjacent to the P site, is where the is where the next amino acid-next amino acid-ttRNA complex RNA complex bbinds inds to the ribosome so that its amino acid to the ribosome so that its amino acid can can bbe attached to the peptide chain. e attached to the peptide chain.
• The E site is where the discharged The E site is where the discharged tRNA is transiently bound before it tRNA is transiently bound before it leaves the ribosome.leaves the ribosome.
prof. azaprof. aza
prof. azaprof. aza
•This large subunit is believed to This large subunit is believed to have have three binding sites called three binding sites called the P (peptidthe P (peptidyyl), A (acceptor) and l), A (acceptor) and E (exit) sites. E (exit) sites.
• It attaches itself to the smaller It attaches itself to the smaller subunit so that its P site is subunit so that its P site is aligned with the methionine-tRNA aligned with the methionine-tRNA complex bound to the mRNA. complex bound to the mRNA.
•This P site is This P site is where the where the growing protein will be bound growing protein will be bound to the ribosometo the ribosome. .
prof. azaprof. aza
• The A site, which is thought to be The A site, which is thought to be adjacent to the P site, adjacent to the P site, is where the is where the next amino acid-tRNA complex binds next amino acid-tRNA complex binds to the ribosome so that its amino acid to the ribosome so that its amino acid can be attached to the peptide chaincan be attached to the peptide chain. .
• The E site is where The E site is where the discharged the discharged tRNA is transiently tRNA is transiently bbound before it ound before it leaves the ribosomeleaves the ribosome
prof. azaprof. aza
prof. azaprof. aza
33.. Elongation Elongation•Elongation is the formation of Elongation is the formation of
the peptide chain of the the peptide chain of the protein protein by a stepwise by a stepwise repetitive process.repetitive process.
•A great deal is known about A great deal is known about the nature of this process the nature of this process bbut ut its exact mechanism is still not its exact mechanism is still not fully understood. fully understood.
•The process of elongation is best The process of elongation is best explained by the use of a explained by the use of a hypothetical example. hypothetical example.
•Suppose that the sequence of Suppose that the sequence of codons, including the start codons, including the start codon, is codon, is AUGAUGUUGUUGGCUGCUGGAGGA.. etc.. etc
prof. azaprof. aza
•The elongation process starts The elongation process starts with with the methionine-tRNA bound the methionine-tRNA bound to the AUG codon of the mRNA to the AUG codon of the mRNA (Figure 14). (Figure 14).
•Because the second codon is Because the second codon is UUG UUG the second amino acid in the second amino acid in the polypetide chain will be the polypetide chain will be leucine. leucine.
prof. azaprof. aza
•This amino acid is transported This amino acid is transported by by a tRNA molecule with the a tRNA molecule with the anticodon AAC anticodon AAC because this is the because this is the only anticodon that matches the only anticodon that matches the UUG codon on the mRNA strand. UUG codon on the mRNA strand.
•The leucine- tRNA complex ‘docks’ The leucine- tRNA complex ‘docks’ on the UUG codon of the mRNA on the UUG codon of the mRNA and and binds to the A site. binds to the A site.
prof. azaprof. aza
prof. azaprof. aza
• This docking and binding is believed This docking and binding is believed to involve rito involve ribbosome proteins, osome proteins, referred to as elongation factors, and referred to as elongation factors, and energy supplied by the hydrolysis of energy supplied by the hydrolysis of guanosine triphosphate (GTP) to guanosine triphosphate (GTP) to guanosine diphosphate (GDP). guanosine diphosphate (GDP).
• Once the leucine-tRNA has occupied Once the leucine-tRNA has occupied the A site the methionin is linked to the A site the methionin is linked to the leucine by means of a peptide the leucine by means of a peptide link link whose carbonyl group originates whose carbonyl group originates from the methionine. from the methionine.
• TThis rhis reeaction is catalysed by the action is catalysed by the appropriate transferase. appropriate transferase.
• It leaves the tRNA on the P site It leaves the tRNA on the P site empty and produces an empty and produces an (NH2)-(NH2)-Met-Leu-tRNA complex Met-Leu-tRNA complex at the A at the A sitesite. .
prof. azaprof. aza
•The empty tRNA is discharged The empty tRNA is discharged through the through the EE site and at the same site and at the same time time the complete ribosome the complete ribosome moves along the mRNA in the 5’ to moves along the mRNA in the 5’ to 3’ 3’ direction so that the direction so that the dipeptide-dipeptide-tRNA complex moves from the A tRNA complex moves from the A site to the P sitesite to the P site. .
prof. azaprof. aza
•This process is known as This process is known as translocationtranslocation. .
• It is poorly understood but It is poorly understood but it leaves it leaves the A site empty and able to the A site empty and able to receive the next amino acid tRNA receive the next amino acid tRNA complex.complex.
•The whole process is then repeated The whole process is then repeated in order to add the next amino acid in order to add the next amino acid residue to peptide chain. residue to peptide chain.
prof. azaprof. aza
• Because the next mRNA codon in our Because the next mRNA codon in our hypothetical example is (hypothetical example is (GCUGCU) this ) this amino acid will be amino acid will be alaninealanine (see Table (see Table 10. l). Subsequent amino acids are 10. l). Subsequent amino acids are added in a similar way, the sequence added in a similar way, the sequence of amino acid residues in tof amino acid residues in thehe chain chain being control led by the order of the being control led by the order of the codons in the mRNA.codons in the mRNA.
prof. azaprof. aza
• It is poorly understood but it It is poorly understood but it leaves the A site empty and able leaves the A site empty and able to receive the next amino acid to receive the next amino acid tRNA complex. The whole tRNA complex. The whole process is then repeated in order process is then repeated in order to add the next amino acid to add the next amino acid residue to peptide chain . residue to peptide chain .
prof. azaprof. aza
• Figure 13Figure 13.. A diagrammatic representation of A diagrammatic representation of the process of elongation in protein synthesisthe process of elongation in protein synthesis
prof. azaprof. aza
prof. azaprof. aza
10.4 10.4 TerminationTermination
•The elongation process continues The elongation process continues until a stop codon is reacheduntil a stop codon is reached..
• ThisThis codon cannot accept an codon cannot accept an amino acid-tRNA complex and so amino acid-tRNA complex and so the synthesis sthe synthesis sttops. ops.
•At this point the peptide-tRN At this point the peptide-tRN chain occupies a P site and chain occupies a P site and the A the A site is empty. site is empty.
•The stop codon of the mRNA is The stop codon of the mRNA is recognised by proteins know as recognised by proteins know as release factorsrelease factors, which promote , which promote the release of the protein from the release of the protein from the ribosome. the ribosome.
prof. azaprof. aza
•The mechanism by which this The mechanism by which this happens is not fully understood happens is not fully understood but they are believed convert the but they are believed convert the transferase responsible for transferase responsible for peptide synthesis into a peptide synthesis into a hydrolase, hydrolase, which catalyses which catalyses hydrolysis of the ester group hydrolysis of the ester group linking the polypeptide to its linking the polypeptide to its tRNA. tRNA.
prof. azaprof. aza
•Once released, the protein is Once released, the protein is folded into its characteristic folded into its characteristic shapeshape. often under the . often under the direction of molecular direction of molecular chaperone protein.chaperone protein.
prof. azaprof. aza
prof. azaprof. aza
11 Protein Synthesis in Prokaryotic and 11 Protein Synthesis in Prokaryotic and Eukaryotic CellsEukaryotic Cells
• The general sequence of events The general sequence of events protein synthesis is similar for both protein synthesis is similar for both eukaryotic and pro prokaryotic cells. eukaryotic and pro prokaryotic cells.
• In both cases In both cases the hydrolysis of GDP the hydrolysis of GDP to GDP is the source of energy for to GDP is the source of energy for many of the processes involvedmany of the processes involved..
• However, the structures of However, the structures of prokaryotic and eukaryotic prokaryotic and eukaryotic ribosomes are differentribosomes are different
•For example, the ribosomes of For example, the ribosomes of prokaryotic cells of bacteria are prokaryotic cells of bacteria are made up made up of 50Sof 50S (see Apendix 3) (see Apendix 3) and 30S rRNA and 30S rRNA subunits whereas subunits whereas the ribosthe ribosoomes of mammalian mes of mammalian eukaryotic cells consist ,of eukaryotic cells consist ,of 60S 60S and 40S rRNA and 40S rRNA subunits. subunits.
prof. azaprof. aza
•The differences between the The differences between the ribosomes of prokaryotic and ribosomes of prokaryotic and eukaryotic ribosomes eukaryotic ribosomes are the are the basis of the selective action of basis of the selective action of some antibioticssome antibiotics . .
prof. azaprof. aza
prof. azaprof. aza
11.111.1.. Prokaryotic CeLLs Prokaryotic CeLLs•The first step in protein synthesis The first step in protein synthesis
is the correct alignment of mRNA is the correct alignment of mRNA on the small subunit of the on the small subunit of the ribosome. ribosome.
• In prokaryotic cells this alignment In prokaryotic cells this alignment is believed to be due to binding is believed to be due to binding by base pairing by base pairing between bases at between bases at the 3’ end of the rRNA of the the 3’ end of the rRNA of the ribosome and bases at the 5’ end ribosome and bases at the 5’ end of the mRNAof the mRNA..
• This ensures the correct alignment of This ensures the correct alignment of the the AUG anticodon of the mRNA with AUG anticodon of the mRNA with the P site of the ribosomethe P site of the ribosome. .
• The mRNA sequence of bases The mRNA sequence of bases responsible for this binding occurs as responsible for this binding occurs as part of the upstream (5’ terminal part of the upstream (5’ terminal end) section of the strand before the end) section of the strand before the start codon. start codon.
prof. azaprof. aza
prof. azaprof. aza
• This sequence is often known as the This sequence is often known as the Shine-Dalgarno sequenceShine-Dalgarno sequence after its after its discovers. Shine-Dalgarno sequences discovers. Shine-Dalgarno sequences vary in length and base sequence vary in length and base sequence (Figure 10.15).(Figure 10.15).
• The initiating tRNA in prokaryotic The initiating tRNA in prokaryotic cells is a specific methionine-tRNA cells is a specific methionine-tRNA known as known as tRNAtRNAff
MetMet ,which is able to ,which is able to read the start codon AUG but not read the start codon AUG but not when it is part of the elongation when it is part of the elongation sequence. tRNAsequence. tRNAff
MetMet is unique in that is unique in that the methionine it carries is usually in the methionine it carries is usually in the form of its N-formyl derivative.the form of its N-formyl derivative.
prof. azaprof. aza
Figure 10.15. Examples of Shine-Dalgarno sequences (bold larger type) of mRNA recognised by Escherichia coli ribosomes. These sequences lie about 10 nucleotides upstream of the AUG start codon for the specified protein.
prof. azaprof. aza
When AUG is part of the elongation sequence methionine is added to the growing protein by a different transfer RNA known as tRNAtRNAmm
MetMet, which also has the anticodon UAC.
• However, tRNAmMet cannot initiate However, tRNAmMet cannot initiate protein synthesis. Elongation follows protein synthesis. Elongation follows the general mechanism for protein the general mechanism for protein synthesis (see section 10.9). synthesis (see section 10.9).
• It requires a group of proteins known It requires a group of proteins known as elongation factors and energy as elongation factors and energy supplied by the hydrolysis of GTP to supplied by the hydrolysis of GTP to GDP. Termination normally involves GDP. Termination normally involves three release factors.three release factors.
prof. azaprof. aza
prof. azaprof. aza
•Experimental work has shown Experimental work has shown that an mRNA strand actively that an mRNA strand actively ssyynthesinthesizzing proteins still have ing proteins still have several ribosomes attached to it several ribosomes attached to it at different places along its at different places along its length. These multiple ribosome length. These multiple ribosome structures are referred to as structures are referred to as polyribosomes or polysomes. polyribosomes or polysomes.
•The polysomes of prokaryotic The polysomes of prokaryotic cells can contain up to 10 cells can contain up to 10 ribosomes at any one lime. Each ribosomes at any one lime. Each of these ribosomes will be of these ribosomes will be simultaneously producing the simultaneously producing the same polypeptide or protein; same polypeptide or protein;
prof. azaprof. aza
•the further the ribosome has the further the ribosome has moved along the mRNA, the moved along the mRNA, the longer the polypeptide chain. The longer the polypeptide chain. The process resembles the assembly process resembles the assembly line in a factory. Each mRNA line in a factory. Each mRNA strand can in its lifetime produce strand can in its lifetime produce up to 300 protein molecules.up to 300 protein molecules.
prof. azaprof. aza
prof. azaprof. aza
10 amino acid residues are 10 amino acid residues are added..added..
• In prokaryotic but not eukaryotic In prokaryotic but not eukaryotic cells (see section 4.1), ribosomes are cells (see section 4.1), ribosomes are found in association with DNA. found in association with DNA.
• This is believed to he due to the This is believed to he due to the ribosome binding to the mRNA as it ribosome binding to the mRNA as it is produced by transcription from the is produced by transcription from the DNA. DNA.
•Furthermore, these ribosomes Furthermore, these ribosomes have been shown to start have been shown to start producing the polypeptide chain producing the polypeptide chain of their designated protein of their designated protein before transcription is complete. before transcription is complete.
prof. azaprof. aza
•This means that in bacteria protein This means that in bacteria protein synthesis can be very rapid and in synthesis can be very rapid and in some cases faster than some cases faster than transcription. It has been reported transcription. It has been reported that in some bacteria an average that in some bacteria an average of 10 amino acid residues are of 10 amino acid residues are added to the peptide chain ever added to the peptide chain ever secondsecond
prof. azaprof. aza
prof. azaprof. aza
11.211.2.. Eukaryotic Cells Eukaryotic Cells
•The initiation of protein synthesis The initiation of protein synthesis in eukaryotic cells follows a in eukaryotic cells follows a different route from that found in different route from that found in prokaryotic cells although it still prokaryotic cells although it still uses a methionine-tRNA to start uses a methionine-tRNA to start the process. the process.
•Eukaryotic mRNAs has no Shine-Eukaryotic mRNAs has no Shine-Dalgarno sequences but are Dalgarno sequences but are characterised by a 7-methyl GTP characterised by a 7-methyl GTP unit at the 5’ end of the mRNA unit at the 5’ end of the mRNA strand and a polyadenosine strand and a polyadenosine nucleotide tail at the 3’ end of nucleotide tail at the 3’ end of the strand (Figure 10.16).the strand (Figure 10.16).
prof. azaprof. aza
prof. azaprof. aza
prof. azaprof. aza
• In eukaryotic cells, the initiating In eukaryotic cells, the initiating tRNA is a unique form of the tRNA is a unique form of the activated methionine- tRNA activated methionine- tRNA (tRNA(tRNAii MetMet). However, unlike in the ). However, unlike in the case of prokaryotic cells, the case of prokaryotic cells, the methionine residue it carries is methionine residue it carries is not formylated. not formylated.
•The initiating process is started by The initiating process is started by this tRNAi Met binding to the 40S this tRNAi Met binding to the 40S subunit of the ribosome to form the subunit of the ribosome to form the so-called preinitiation complex, the so-called preinitiation complex, the process requiring the formation of a process requiring the formation of a complex between tRNAi Met , complex between tRNAi Met , various eukaryotic initiation factors various eukaryotic initiation factors (elFs) and GTP. (elFs) and GTP.
prof. azaprof. aza
•At this point the mRNA binds to the 40S At this point the mRNA binds to the 40S preinitiation complex. This binding process preinitiation complex. This binding process is believed to involve a number of is believed to involve a number of eukaryotic initiation factors and energy eukaryotic initiation factors and energy supplied by the conversions of GTP to GDP supplied by the conversions of GTP to GDP and ATP to ADP. Once the mRNA has and ATP to ADP. Once the mRNA has bound to the preinitiation complex the 60S bound to the preinitiation complex the 60S subunit recombines with the 40S unit to subunit recombines with the 40S unit to form the initiation complex (Figure 10.17).form the initiation complex (Figure 10.17).
prof. azaprof. aza
•Once the mRNA has bound to Once the mRNA has bound to the preinitiation complex the the preinitiation complex the 60S subunit recombines with 60S subunit recombines with the 40S unit to form the the 40S unit to form the initiation complex (Figure initiation complex (Figure 10.17).10.17).
prof. azaprof. aza
•The initiating process is started The initiating process is started by this tRNAi Met binding to the by this tRNAi Met binding to the 40S subunit of the ribosome to 40S subunit of the ribosome to form the so-called preinitiation form the so-called preinitiation complex, the process requiring complex, the process requiring the formation of a complex the formation of a complex between tRNAi Met , various between tRNAi Met , various eukaryotic initiation factors (elFs) eukaryotic initiation factors (elFs) and GTP. and GTP.
prof. azaprof. aza
prof. azaprof. aza
•The absence of the Shine-The absence of the Shine-Dalgarno sequence means that Dalgarno sequence means that an alternative mechanism must an alternative mechanism must he available to align the first he available to align the first AUG codon of the mRNA with the AUG codon of the mRNA with the P site of the ribosome. This P site of the ribosome. This mechanism is believed to direct mechanism is believed to direct the preinitiation complex to the the preinitiation complex to the first AUG codon of the mRNA.first AUG codon of the mRNA.
• Elongation in eukaryotic ribosomes Elongation in eukaryotic ribosomes follows the general mechanism for follows the general mechanism for protein synthesis (see section 10.10.3) protein synthesis (see section 10.10.3) but involves different factors and but involves different factors and proteins from those utilised by proteins from those utilised by prokaryotic ribosomes. Termination only prokaryotic ribosomes. Termination only requires one release factor, unlike in requires one release factor, unlike in prokaryotic ribosomes-where three prokaryotic ribosomes-where three release factors are usually required.release factors are usually required.
prof. azaprof. aza
• Elongation in eukaryotic ribosomes Elongation in eukaryotic ribosomes follows the general mechanism for follows the general mechanism for protein synthesis (see section 10.10.3) protein synthesis (see section 10.10.3) but involves different factors and but involves different factors and proteins from those utilised by proteins from those utilised by prokaryotic ribosomes. prokaryotic ribosomes.
• Termination only requires one release Termination only requires one release factor, unlike in prokaryotic ribosomes-factor, unlike in prokaryotic ribosomes-where three release factors are usually where three release factors are usually required.required.
prof. azaprof. aza
•Termination Termination only requires one only requires one release factor,release factor, unlike in unlike in prokaryotic ribosomes-where prokaryotic ribosomes-where three release factors are three release factors are usually required.usually required.
prof. azaprof. aza
prof. azaprof. aza
Figure 10.17. An outline of the formation of the protein synthesis initiation complex by the ribosomes of eukaryotic cells.
prof. azaprof. aza
12. Bacterial Protein 12. Bacterial Protein Synthesis InhibitorsSynthesis Inhibitors
Antimicrobials)Antimicrobials)• Many protein inhibitors inhibit protein Many protein inhibitors inhibit protein
synthesis in both prokaryotic and synthesis in both prokaryotic and eukaryotic cells (Table 10.2). eukaryotic cells (Table 10.2).
• This inhibition can take place at any This inhibition can take place at any stage in protein synthesis. stage in protein synthesis.
• However, some inhibitors have a However, some inhibitors have a specific action in that they inhibit specific action in that they inhibit protein synthesis in prokaryotic cells protein synthesis in prokaryotic cells but not in eukaryotic cells, or vice but not in eukaryotic cells, or vice versa. versa.
• Consequently, a number of useful drugs Consequently, a number of useful drugs have been discovered that will inhibit have been discovered that will inhibit protein synthesis in bacteria protein synthesis in bacteria but but either either have no effect or a very much reduced have no effect or a very much reduced effect on protein synthesis in mammalseffect on protein synthesis in mammals..
• The structures and activities of the The structures and activities of the drugs that inhibit protein synthesis are drugs that inhibit protein synthesis are quite diverse. quite diverse.
prof. azaprof. aza
•Consequently, only a few of the Consequently, only a few of the more commonly used drugs and more commonly used drugs and structurally related compounds structurally related compounds will be discussed in greater detail will be discussed in greater detail in this section.in this section.
prof. azaprof. aza
prof. azaprof. aza
Table 10.2. Examples of drugs that inhibit protein synthesis.
prof. azaprof. aza
12.112.1.. Aminoglycosides Aminoglycosides
• Streptomycin (Figure 10.15) is a Streptomycin (Figure 10.15) is a member of a group of compounds member of a group of compounds known as aminoglycosides. known as aminoglycosides.
• These compounds have structures in These compounds have structures in which aminowhich amino sugar residues in the sugar residues in the form of mono- or polysaccharides are form of mono- or polysaccharides are attached to a substituted attached to a substituted 1 ,3-1 ,3-diaminocyclohexane ring by modified diaminocyclohexane ring by modified glycosidic type linkages. glycosidic type linkages.
•The ring is either The ring is either streptidinestreptidine (streptomycin) or (streptomycin) or deoxy deoxy streptamine streptamine (kanamycin, (kanamycin, neomycin, gentamicin and neomycin, gentamicin and tobramycin).tobramycin).
prof. azaprof. aza
prof. azaprof. aza
Figure 10.18. The structures of (a) streptomycin and (b) neomiycin C.
prof. azaprof. aza
•Streptomycin Streptomycin iis as the first s as the first aminoglycoside discovered aminoglycoside discovered (Schatz and co-workers. 1 44) (Schatz and co-workers. 1 44) from cultures of the soil from cultures of the soil ActinomyceteActinomycetess Streptomyces Streptomyces griseus. griseus.
• It acts by interfering with the It acts by interfering with the initiation of protein synthesis in initiation of protein synthesis in bacteria. bacteria.
• The binding of streptomycin to the The binding of streptomycin to the 30S ribosome inhibits initiation30S ribosome inhibits initiation and and also causes some amino acid-tRNA also causes some amino acid-tRNA complexes to complexes to misread the mRNA misread the mRNA codonscodons..
prof. azaprof. aza
•This results in This results in the insertion of the insertion of incorrect amino acid residues incorrect amino acid residues into the protein chain, which into the protein chain, which usually leads to the death of the usually leads to the death of the bacteriabacteria. .
prof. azaprof. aza
•The mode of action of the other The mode of action of the other aminoglycosides has been aminoglycosides has been assumed assumed to follow the same to follow the same pattern pattern even though most of the even though most of the investigations into the investigations into the mechanism of the antibacterial mechanism of the antibacterial action of the aminoglycocides action of the aminoglycocides have been carried out have been carried out
prof. azaprof. aza
prof. azaprof. aza
•The clinically used The clinically used aminoglycosides have structures aminoglycosides have structures closely related to that of closely related to that of streptomycin. streptomycin.
•They are They are essentially broad-essentially broad-spectrum antibiotics although spectrum antibiotics although the are normally used to treat the are normally used to treat serious Gram-negativeserious Gram-negative bacterial bacterial infections (see section 4.2.5.1). infections (see section 4.2.5.1).
• Aminoglycosidic drugs Aminoglycosidic drugs are very water are very water soluble. soluble. They are usually They are usually administered as their water-soluble administered as their water-soluble inorganic salts but their polar nature inorganic salts but their polar nature means that the’ are means that the’ are poorly absorbed poorly absorbed when administered orally.when administered orally.
• Once in the body they are easily Once in the body they are easily distributed into most body fluids. distributed into most body fluids.
prof. azaprof. aza
• However, their polar nature means However, their polar nature means that that they do not easily penetrate the they do not easily penetrate the central nervous system (CNS), bone, central nervous system (CNS), bone, fatty and connective tissuefatty and connective tissue. .
• Moreover, aminoglycosides Moreover, aminoglycosides tend to tend to concentrate in the kidney concentrate in the kidney where thewhere theyy are excreted by glomerular filtration.are excreted by glomerular filtration.
prof. azaprof. aza
prof. azaprof. aza
Figure 19. Kanamycin.
prof. azaprof. aza
• Aminoglycoside-drug-resistant Aminoglycoside-drug-resistant strains of bacteria are not recognised strains of bacteria are not recognised as a serious medical problem. as a serious medical problem.
• They arise because dominant They arise because dominant bacteria strains have emerged bacteria strains have emerged that that possess enzymes that effectively possess enzymes that effectively inactivate the druginactivate the drug. .
•These enzymes act by catalysing These enzymes act by catalysing the the acylationacylation,, phosphorylation phosphorylation and adenylation of the drug and adenylation of the drug (see (see section 6.13). This results in the section 6.13). This results in the formation of inactive drug formation of inactive drug derivatives.derivatives.
prof. azaprof. aza
• The activity of the aminoglycosides The activity of the aminoglycosides is is related to the nature of their ring related to the nature of their ring substisubstittuents. uents.
• Consequently, it is convenient to Consequently, it is convenient to discuss this activity in relation to the discuss this activity in relation to the changes in the substituents of individual changes in the substituents of individual rings but, in view of the diversity of the rings but, in view of the diversity of the structures of aminoglycosides, it is structures of aminoglycosides, it is difficult to identify common trends. difficult to identify common trends.
prof. azaprof. aza
• As a result, this discussion will As a result, this discussion will bbe e largely limited to kanamycin (Figure largely limited to kanamycin (Figure 10.19). 10.19).
• However, the same trends are often However, the same trends are often true for other aminoglycosides whose true for other aminoglycosides whose structures consist of three rings, structures consist of three rings, including a central deoxystreptamine including a central deoxystreptamine residue.residue.
prof. azaprof. aza
prof. azaprof. aza
• Changing the nature of the amino Changing the nature of the amino substituents at positions 2’ and 6’ of substituents at positions 2’ and 6’ of ring I has the greatest effect on ring I has the greatest effect on activity. activity.
• For example, For example, kanamycin A, which has a kanamycin A, which has a hydroxy group at position 2’, and hydroxy group at position 2’, and kanamycin C, which has a hydroxy kanamycin C, which has a hydroxy group at position 6’, are both less group at position 6’, are both less active than kanamycin B, active than kanamycin B, which has which has amino groups at the 2’ and 6’ positions. amino groups at the 2’ and 6’ positions.
•However, the removal of one or However, the removal of one or both of the hydroxy groups at both of the hydroxy groups at positions 3’ and 4’ does not have positions 3’ and 4’ does not have any effect on the potency any effect on the potency of the of the kanamycins.kanamycins.
prof. azaprof. aza
prof. azaprof. aza
•Modifications to ring II (the Modifications to ring II (the deoxystreptamine ring) gdeoxystreptamine ring) greatly reatly reduce the potency of the reduce the potency of the kanamycins. kanamycins.
•However, N-acylation and However, N-acylation and alkylation of the amino group at alkylation of the amino group at position I can give compounds position I can give compounds with some activitwith some activityy
•For example, acylation of For example, acylation of kanamycin A gives 1-N-(L (-)-4- kanamycin A gives 1-N-(L (-)-4- amino-2-hydroxybutyryl) amino-2-hydroxybutyryl) kanamycin A (amikacin), which kanamycin A (amikacin), which has a potency of about 50% of has a potency of about 50% of that of kanamycin A (Figure that of kanamycin A (Figure 10.20). 10.20).
prof. azaprof. aza
• In spite of this, amikacin is a useful In spite of this, amikacin is a useful drug for treating some strains of drug for treating some strains of Gram-negative bacteria because it is Gram-negative bacteria because it is resistant to deactivation by bacterial resistant to deactivation by bacterial enzymes. Similarly, 1-N-enzymes. Similarly, 1-N-ethylsisomicin (netilmicin) is as ethylsisomicin (netilmicin) is as potent as its parent aminoglvcoside potent as its parent aminoglvcoside sisomicin.sisomicin.
prof. azaprof. aza
prof. azaprof. aza
• Changing the substituents of ring III Changing the substituents of ring III does not usually have such a great does not usually have such a great effect on the potency of the drug as effect on the potency of the drug as similar changes in ring I and II.similar changes in ring I and II.
• For example, removal of the For example, removal of the 2” 2” hydroxy group of gentamicin results hydroxy group of gentamicin results in a in a significant drop in activity. significant drop in activity.
• However, replacement of the 2” However, replacement of the 2” hydroxhydroxy y group of gentamicin (Figure group of gentamicin (Figure 21) by amino groups gives the highly 21) by amino groups gives the highly active seldomycins.active seldomycins.
prof. azaprof. aza
prof. azaprof. aza
Figure 20. An out!ine of the chemistry involved in the synthesis of the antibiotics amikacin and netilmicin. Cbz is frequently used as a protecting group for amines because it is easily removed by hydrogenation.
prof. azaprof. aza
Figure 10.21. The structures of gentamicin.
prof. azaprof. aza
12.2. Chloramphenicol12.2. ChloramphenicolChloramphenicol was first isolated from the microorganism Streptomyces venezuela by Ehrlich and co-workers in 1947. It is a broad-spectrum antibiotic whose structure contains two asymmetric centres. However. only the D(-)-threo form is active.
prof. azaprof. aza
prof. azaprof. aza
•Chloramphenicol can cause Chloramphenicol can cause serious side effects and so it is serious side effects and so it is recommended that it is only recommended that it is only used for specific infections. used for specific infections. It is It is often administered as its often administered as its palmitate in order to mask its palmitate in order to mask its bitter taste.bitter taste.
• The free drug is liberated from this The free drug is liberated from this ester by hydrolysis in the duodenum ester by hydrolysis in the duodenum chloramphenicol has a poor water chloramphenicol has a poor water solubility (2.5 gsolubility (2.5 g//dm-3 ) and So it is dm-3 ) and So it is sometimes administered in the form sometimes administered in the form of its of its sodium hemisuccinate salt (see sodium hemisuccinate salt (see section 3.7.4.2), which acts as a section 3.7.4.2), which acts as a prodrug.prodrug.
prof. azaprof. aza
prof. azaprof. aza
•ChChlloramphenicol acts by oramphenicol acts by inhibiting the elongation stage in inhibiting the elongation stage in protein synthesis in prokaryotic protein synthesis in prokaryotic cells. cells.
• It binds reversibly to the 50S It binds reversibly to the 50S ribosome subunit and is thought ribosome subunit and is thought to prevent the binding of the to prevent the binding of the aminoacyl-tRNA complex to aminoacyl-tRNA complex to the ribosome. the ribosome.
• However, its precise mode of action However, its precise mode of action is not understood.is not understood.
• Investigation of the activity of Investigation of the activity of analogues of chloramphenicol analogues of chloramphenicol showed showed that activity requires a para-that activity requires a para-electron-withdrawing group. electron-withdrawing group.
prof. azaprof. aza
• However, substituting the nitro group However, substituting the nitro group with other electron-withdrawing groups with other electron-withdrawing groups gave compounds with a reduced gave compounds with a reduced activity. Furthermore, modification of activity. Furthermore, modification of the side chain, with the exception of the side chain, with the exception of the difluoro derivative, gave the difluoro derivative, gave compounds that had a lower activity compounds that had a lower activity than chloramphenicol (Table 10.3). than chloramphenicol (Table 10.3).
prof. azaprof. aza
• These observations suggest that These observations suggest that D(-)-threo chloramphenicol has the D(-)-threo chloramphenicol has the optimum structure of those tested for optimum structure of those tested for activity.activity.
prof. azaprof. aza
• Investigation of the activity of Investigation of the activity of analogues of chloramphenicol analogues of chloramphenicol showed that activity requires a para-showed that activity requires a para-electron-withdrawing group. electron-withdrawing group. However, However, substituting the nitro group substituting the nitro group with other electron-withdrawing with other electron-withdrawing groups gave compounds with a groups gave compounds with a reduced activityreduced activity. .
prof. azaprof. aza
• Furthermore, modification of the side Furthermore, modification of the side chain, with the exception of the chain, with the exception of the difluoro derivativedifluoro derivative, gave compounds , gave compounds that had a lower activity than that had a lower activity than chloramphenicolchloramphenicol (Table 10.3). (Table 10.3).
• These observations suggest that These observations suggest that D(-)-threo chloramphenicol has the D(-)-threo chloramphenicol has the optimum structure of those tested for optimum structure of those tested for activity.activity.
prof. azaprof. aza
prof. azaprof. aza
Table 10.3. The activity against Escherichia coli of some analogues chloramphenicol relative to chloramphenicol.
prof. azaprof. aza
12.3 TetracycLines12.3 TetracycLines
• Tetracyclines are a family of natural Tetracyclines are a family of natural and semisynthetic antibiotics and semisynthetic antibiotics isolated from various Streptomyces isolated from various Streptomyces species, the first member of the species, the first member of the group chlortetracycline being group chlortetracycline being obtained in 1945 by Duggar from obtained in 1945 by Duggar from Streptomyces aureofaciens.Streptomyces aureofaciens. A A number of highly active number of highly active semisynthetic analogues have also semisynthetic analogues have also been prepared from the naturally been prepared from the naturally occurring compounds (Table 10.4). occurring compounds (Table 10.4).
prof. azaprof. aza
Table 10.4.The structures of the tetracyclines.
• The tetracvclines are The tetracvclines are a broad-spectrum a broad-spectrum group of antibiotics active against group of antibiotics active against many Gram-positive and Gram-many Gram-positive and Gram-negative negative bacteria, rickettsiae, bacteria, rickettsiae, mycoplasmas, chlamydiae and some mycoplasmas, chlamydiae and some protozoa that cause malaria. A number protozoa that cause malaria. A number of the natural and semisynthetic of the natural and semisynthetic compounds are in current medical use.compounds are in current medical use.
prof. azaprof. aza
prof. azaprof. aza
•The structures of the The structures of the tetracyclines are based on four-tetracyclines are based on four-ring system. Their structures are ring system. Their structures are complicated by the presence of complicated by the presence of up to six chiral carbons in the up to six chiral carbons in the fused-ring systemfused-ring system. These . These normally occur at positions 4, 4a, normally occur at positions 4, 4a, 5, 5a, 6 and 12a, depending on 5, 5a, 6 and 12a, depending on the symmetry of the structure. the symmetry of the structure.
•The configurations of these The configurations of these centres in the active compounds centres in the active compounds have been determined by X-ray have been determined by X-ray crystallography (Table 10.4). This crystallography (Table 10.4). This technique has also confirmed technique has also confirmed that that C1 to C3 and C11 to C12 C1 to C3 and C11 to C12 were conjugated structures.were conjugated structures.
prof. azaprof. aza
prof. azaprof. aza
Table 10.4.The structures of the tetracyclines.
prof. azaprof. aza
•Tetracyclines are amphoteric, Tetracyclines are amphoteric, forming salts with acids and forming salts with acids and bases. bases. They normally exhibit They normally exhibit three pKa. ranges of 2.5 —3.4 three pKa. ranges of 2.5 —3.4 (pKa1 ), 7.2-7.5 (pKa2.) and 9.1(pKa1 ), 7.2-7.5 (pKa2.) and 9.1—9.7 (pKa3.), —9.7 (pKa3.), the last being the the last being the range for the corresponding range for the corresponding ammonium salts. These values ammonium salts. These values have been assigned by Leeson have been assigned by Leeson and co-workers to the structures and co-workers to the structures shown in Table 10.4. shown in Table 10.4.
• These assignments have been supported These assignments have been supported by the work of Rigler and collegues. by the work of Rigler and collegues. However, the assignments for pKa2, and However, the assignments for pKa2, and pKa3, are opposite to those suggested pKa3, are opposite to those suggested by Stephens and collegues. by Stephens and collegues.
• Tetracyclines also have a strong affinity Tetracyclines also have a strong affinity for metal ions, forming stable chelates for metal ions, forming stable chelates with calcium, magnesium and iron ions. with calcium, magnesium and iron ions.
prof. azaprof. aza
• These chelates are usually soluble in These chelates are usually soluble in water, which accounts for water, which accounts for the poor the poor absorption of tetracyclines in the absorption of tetracyclines in the presence of drugs and foods that presence of drugs and foods that contain these metal ions. contain these metal ions.
• HoweverHowever, , this affinity for metals this affinity for metals appears to play an essential role in appears to play an essential role in the action of tetracthe action of tetracyyclines.clines.
prof. azaprof. aza
prof. azaprof. aza
• Tetracyclines are transported into Tetracyclines are transported into the bacterial cell by passive diffusion the bacterial cell by passive diffusion and active transport. Active transport and active transport. Active transport requires the presence of Mgrequires the presence of Mg22 ions ions and ATP possibly as an energy and ATP possibly as an energy source. source.
• Once in the bacteria, Once in the bacteria, tetracyclines tetracyclines act by preventing protein elongation act by preventing protein elongation by inhibiting the binding of the by inhibiting the binding of the aminoacyl-tRNA to the 30S subunit of aminoacyl-tRNA to the 30S subunit of the prokaryotic ribosome. the prokaryotic ribosome.
• This binding has also been shown to This binding has also been shown to require magnesium ions.require magnesium ions.
prof. azaprof. aza
prof. azaprof. aza
•Tetracyclines also Tetracyclines also penetrate penetrate mammalian cells and bind to mammalian cells and bind to eukaryotic ribosomes. eukaryotic ribosomes.
•However, their affinity for However, their affinity for eukaryotic ribosomes is lower than eukaryotic ribosomes is lower than that for prokaryotic ribosomes and that for prokaryotic ribosomes and so they so they do not achieve a high do not achieve a high enough concentration to disrupt enough concentration to disrupt eukaryotic protein synthesiseukaryotic protein synthesis. .
bacterial resistancebacterial resistance
• Unfortunately, bacterial resistance to Unfortunately, bacterial resistance to tetracyclines is common. It is believed tetracyclines is common. It is believed to involve three distinct mechanisms, to involve three distinct mechanisms, namely: namely: active transport of the drug active transport of the drug out of the bacteria by membrane out of the bacteria by membrane spanning proteins; enzymic oxidation spanning proteins; enzymic oxidation of the drug; and ribosome protectionof the drug; and ribosome protection by chromosomal protein determinants.by chromosomal protein determinants.
prof. azaprof. aza
prof. azaprof. aza
•The structure-activity The structure-activity relationships of tetracyclines relationships of tetracyclines have been extensively have been extensively investigated and reported. investigated and reported.
•Consequently, the following Consequently, the following paragraphs give only a synopsis paragraphs give only a synopsis of these relationships.of these relationships.
•This synopsis only considers This synopsis only considers general changes to both the general changes to both the general structure of the general structure of the tetracyclines (Figure 10.22) and tetracyclines (Figure 10.22) and the substitution patterns of their the substitution patterns of their individual rings.individual rings.
prof. azaprof. aza
prof. azaprof. aza
•Activity in the tetracyclines Activity in the tetracyclines requires four rings with requires four rings with a cis A/B a cis A/B ring fusion. ring fusion.
•Derivatives with three rings are Derivatives with three rings are usually inactive or almost usually inactive or almost inactive. In general, modification inactive. In general, modification to any of the substituent groups to any of the substituent groups in tin thhe positions e positions C-10, C-11, C-C-10, C-11, C-11a, C-l2, C-12a, C-1, C-2, C-3 11a, C-l2, C-12a, C-1, C-2, C-3 and C-4 results in a significant and C-4 results in a significant loss in activity. loss in activity.
•Changes to the 4-dimethylamino Changes to the 4-dimethylamino group also usually reduce activitygroup also usually reduce activity. . This group must have an a-This group must have an a-configuration and partial configuration and partial conversion of this group to its conversion of this group to its β-β-epimeepimer under acidic conditions at r under acidic conditions at room temperature room temperature significantly significantly reduces activity.reduces activity.
prof. azaprof. aza
prof. azaprof. aza
Figure 10.22. General structure activity relationships in the tetracyclines.
prof. azaprof. aza
• In addition, either removal of the a-dimethIn addition, either removal of the a-dimethyylamino lamino group at position 4 or replacement of one or more of group at position 4 or replacement of one or more of its methyl groups by larger alkyl groups also reduces its methyl groups by larger alkyl groups also reduces activity. Ester formation at C-12a gives inactive activity. Ester formation at C-12a gives inactive esters, with the exception of the formyl ester, which esters, with the exception of the formyl ester, which hydrolyses in aqueous solution to the parent hydrolyses in aqueous solution to the parent tetracycline. Alkylation of C-11a also gives rise to a tetracycline. Alkylation of C-11a also gives rise to a loss of activity.loss of activity.
• In addition, either removal of the a-In addition, either removal of the a-dimethdimethyylamino group at position 4 or lamino group at position 4 or replacement of one or more of its methyl replacement of one or more of its methyl groups by larger alkyl groups also groups by larger alkyl groups also reduces activity. reduces activity.
• Ester formation at C-12a gives inactive Ester formation at C-12a gives inactive esters, with the exception of the formyl esters, with the exception of the formyl ester, which hydrolyses in aqueous ester, which hydrolyses in aqueous solution to the parent tetracycline. solution to the parent tetracycline. Alkylation of C-11a also gives rise to a Alkylation of C-11a also gives rise to a loss of activity.loss of activity.
prof. azaprof. aza
• Alkylation of C-11a also gives rise to Alkylation of C-11a also gives rise to a loss of activity.a loss of activity.
prof. azaprof. aza
• Modification of the substituents at Modification of the substituents at positions 5, 5a. 6. 7. 8 and 9 may positions 5, 5a. 6. 7. 8 and 9 may lead to similar or increased activity. lead to similar or increased activity.
• Minor changes to the substituents at Minor changes to the substituents at these positions tend to change the these positions tend to change the pharmacokinetic properties rather pharmacokinetic properties rather than activity (Table 10.5). than activity (Table 10.5).
prof. azaprof. aza
prof. azaprof. aza
Table 10.5. The pharmacokinetic properties of tetracycline hyrochlorides. The values given are representative values only because variations between individuals can be quite large.
•A number of active derivatives A number of active derivatives have been synthesihave been synthesizzed by ed by electrophilic substitution of C-7 electrophilic substitution of C-7 and C-9 but the effect of and C-9 but the effect of introducing suintroducing subbssttituents at C-8 has ituents at C-8 has not been studied because this not been studied because this position is difficult to substitute.position is difficult to substitute.
prof. azaprof. aza
prof. azaprof. aza
1313.. Drugs that Target Nucleic Acids Drugs that Target Nucleic Acids
•Drugs that target DNA and RNA Drugs that target DNA and RNA either inhibit their synthesis or either inhibit their synthesis or act on existing nucleic acid act on existing nucleic acid molecules. Those that inhibit the molecules. Those that inhibit the synthesis of nucleic acids usually synthesis of nucleic acids usually act as either act as either antimetabolites or antimetabolites or enzyme inhibitorsenzyme inhibitors. .
•The drugs that target existing The drugs that target existing nucleic acid molecules can, for nucleic acid molecules can, for convenience be broadly convenience be broadly classified into classified into intercalating intercalating agents, alkylating agents and agents, alkylating agents and chain-cleaving agentschain-cleaving agents..
prof. azaprof. aza
prof. azaprof. aza
• However, it should be realised that However, it should be realised that these classifications are not rigid: these classifications are not rigid: drugs may act by more than one drugs may act by more than one mechanism. Those drugs acting on mechanism. Those drugs acting on existing DNA usually existing DNA usually inhibit inhibit transcription whereas those acting transcription whereas those acting on RNA normally inhibit translation. on RNA normally inhibit translation. In both cases the net result is the In both cases the net result is the prevention or slowing down of cell prevention or slowing down of cell growth and division. growth and division.
• Consequently, the discovery of new Consequently, the discovery of new drugs that target existing DNA and drugs that target existing DNA and RNA is a major consideration when RNA is a major consideration when dedevveloping new drugs for the eloping new drugs for the treatment of cancer (see Appendix 4) treatment of cancer (see Appendix 4) and bacterial and other infections and bacterial and other infections due to microorganisms.due to microorganisms.
prof. azaprof. aza
prof. azaprof. aza
13.1 Antimetabolites13.1 Antimetabolites
• Antimetabolites are compounds that Antimetabolites are compounds that block the normal metabolic pathways block the normal metabolic pathways operating inoperating in cells. cells.
• They act by either They act by either replacing an replacing an endogenous compound endogenous compound in the in the pathway by apathway by a compound whose compound whose incorporation into the system results incorporation into the system results in a product that can no longer playin a product that can no longer play any further part in the pathway, any further part in the pathway, or or inhibiting an enzyme in the metabolic inhibiting an enzyme in the metabolic pathway ipathway in then the cell. cell.
•Both these types of intervention Both these types of intervention inhibit the targeted metabolic inhibit the targeted metabolic pathway to a level that hopefully pathway to a level that hopefully has it significant effect on the has it significant effect on the health of the patient.health of the patient.
prof. azaprof. aza
prof. azaprof. aza
• The structures of antimetabolites are The structures of antimetabolites are usually very similar to those of the usually very similar to those of the normal metabolites used by the cell. normal metabolites used by the cell. Those used to prevent the formation Those used to prevent the formation of DNA may he classified as of DNA may he classified as antifolates, pyrimidine antifolates, pyrimidine antimetabolites and purine antimetabolites and purine antimetabolitesantimetabolites. .
• However, because of the difficult of However, because of the difficult of classifying biologically active classifying biologically active substances (see section 1.6), substances (see section 1.6), antimetabolites that inhibit enzyme antimetabolites that inhibit enzyme action are also classified as enzyme action are also classified as enzyme inhibitors.inhibitors.
prof. azaprof. aza
prof. azaprof. aza
Figure 10.23. (a) The structure of folic acid. In blood, folic acids usually have one glutamate residue. However, in the cell they are converted to polyglutamates. (b) A Fragment of a polyglutamate chain.
prof. azaprof. aza
13.1.113.1.1.. Antifolates Antifolates
• Folic acid (Figure 10.23) is usually Folic acid (Figure 10.23) is usually regarded as the parent of a family of regarded as the parent of a family of naturallv occurring compounds naturallv occurring compounds known as folates. known as folates.
• These folates are widely distributed These folates are widely distributed in food. They differ from folic acid in in food. They differ from folic acid in such ways as the state of reduction such ways as the state of reduction of the pteridine ring and having of the pteridine ring and having carbon units attached to either or carbon units attached to either or both of the N5 and N 10 atoms.both of the N5 and N 10 atoms.
• In the body folates are converted by a two-In the body folates are converted by a two-step process into tetrahvdrofolates (FH4) by step process into tetrahvdrofolates (FH4) by the action of the enzyme dihydrofolate the action of the enzyme dihydrofolate reductase (DHFR). reductase (DHFR). Tetrahydrofolic acid is an Tetrahydrofolic acid is an essential cofactor in the biosynthesis of essential cofactor in the biosynthesis of purines and thymine. which are required for purines and thymine. which are required for DNA synthesis.DNA synthesis.
prof. azaprof. aza
prof. azaprof. aza
• Folic acid antimetabolites have Folic acid antimetabolites have structures that resemble folic acid structures that resemble folic acid (Figure 10.24). (Figure 10.24). They have a stronger They have a stronger affinity for DHFR than folic acid affinity for DHFR than folic acid and act and act by inhibiting this enzyme at both stages by inhibiting this enzyme at both stages in the conversion of folic acid to FH4. in the conversion of folic acid to FH4.
• This has the effect of This has the effect of inhibiting the inhibiting the formation of purines and thymine formation of purines and thymine required for DNA synthesis. required for DNA synthesis.
• This inhibits cell growth, which This inhibits cell growth, which prevents replication and ultimately prevents replication and ultimately leads to cell death.leads to cell death.
• MethotrexateMethotrexate is the only folate is the only folate antimetabolite in clinical use. It is antimetabolite in clinical use. It is distributed to most body fluids but distributed to most body fluids but has a low lipid solubility, which has a low lipid solubility, which means, that means, that does not readilv cross does not readilv cross the blood-brain barrier.the blood-brain barrier.
prof. azaprof. aza
prof. azaprof. aza
Figure 10.24. A comparison of the structures of folic acid antimetabolites with folic acid.
prof. azaprof. aza
• It is transported into cells by the It is transported into cells by the folate transport system and at high folate transport system and at high blood levels an additional second blood levels an additional second transport mechanism comes into transport mechanism comes into operationoperation. .
• Once in the cell it is metabolised to Once in the cell it is metabolised to the polyglutamate, which is retained the polyglutamate, which is retained in the cell for considerable periods of in the cell for considerable periods of time. This is probably due to the time. This is probably due to the polar nature of the polymer. polar nature of the polymer.
• MethotrexateMethotrexate is used to treat a is used to treat a variety of cancers, including head variety of cancers, including head and neck tumours, and, in low doses, and neck tumours, and, in low doses, rheumatoid arthritis. rheumatoid arthritis.
• It can cause vomiting, nausea, oral It can cause vomiting, nausea, oral and gastric ulceration and depression and gastric ulceration and depression of bone marrow, a well as other of bone marrow, a well as other unwanted side effects.unwanted side effects.
prof. azaprof. aza
prof. azaprof. aza
13.1.2 Purine Antimetabolites13.1.2 Purine Antimetabolites• Purine antimetaholites are Purine antimetaholites are
exogenous compounds. such as 6-exogenous compounds. such as 6-mercaptopurine and 6-thioguanine, mercaptopurine and 6-thioguanine, with structures based on the purine with structures based on the purine nucleus (Figure 1O.25).nucleus (Figure 1O.25).
• They inhibit the synthesis of DNA and They inhibit the synthesis of DNA and in some cases RNA by a number ol in some cases RNA by a number ol different mechanisms. For example, different mechanisms. For example, 6-mercaptopurine is metabolised to 6-mercaptopurine is metabolised to the ribonucleotide 6-thioguanosine-the ribonucleotide 6-thioguanosine-5’-pltosphate. 5’-pltosphate.
prof. azaprof. aza
Figure 10.25. Examples of purine antimetaholites. The purine nucleus, on which the structures o the antimetabolites and the endogenous compounds they replace are based, is shown in square brackets.
• This exogenous nucleotide inhibits This exogenous nucleotide inhibits several pathways br the biosynthesis several pathways br the biosynthesis of endogenous purine nucleotides. In of endogenous purine nucleotides. In contrast. 6-thioguanine is converted contrast. 6-thioguanine is converted in the cell to the ribonucleotide 6-in the cell to the ribonucleotide 6-thioinosine-5’-phnsphate. thioinosine-5’-phnsphate.
prof. azaprof. aza
• This ribonucleotide disrupts DNA This ribonucleotide disrupts DNA synthesis synthesis by being incorporated into by being incorporated into the structure of DNA as a false nucleic the structure of DNA as a false nucleic acid. acid.
• Resistance to these two drugs arises Resistance to these two drugs arises because of a loss of the posphorybosil because of a loss of the posphorybosil transferase required for the formation transferase required for the formation of their ribonucleotides.of their ribonucleotides.
prof. azaprof. aza
prof. azaprof. aza
13.1.3 Pyrimidine Antimetabolltes13.1.3 Pyrimidine Antimetabolltes
• These are antirnetaholites whose structures closely resenThle those These are antirnetaholites whose structures closely resenThle those of the endouenous pvrimidine bases (Figure l0.26a).They usually act of the endouenous pvrimidine bases (Figure l0.26a).They usually act by inhibiting one or more of the enzymes that areby inhibiting one or more of the enzymes that are required for DNA required for DNA synthesis. synthesis.
• For example, fluorouracil is metabolised by the same metabolic For example, fluorouracil is metabolised by the same metabolic pathway as uracil to 5-fluoro-2’-deoxyuridylic acid (FUdRP). FUdRP pathway as uracil to 5-fluoro-2’-deoxyuridylic acid (FUdRP). FUdRP inhibits the enzyme thyniidylate synthetase. which in its normal role inhibits the enzyme thyniidylate synthetase. which in its normal role is responsible for the transfer (if a meth\ I group from the coenzyme is responsible for the transfer (if a meth\ I group from the coenzyme rnelhylenetetrahydrofolic acid (MeFI 14) to the C5 atom of rnelhylenetetrahydrofolic acid (MeFI 14) to the C5 atom of deoxyuridylic acid (UdRP). The presence of the unreactive C5 F bond deoxyuridylic acid (UdRP). The presence of the unreactive C5 F bond in FUdRP blocks this methylation, which prevents the formation of in FUdRP blocks this methylation, which prevents the formation of deoxythymidylic acid (TdRP) and its subseciuent incorporation into deoxythymidylic acid (TdRP) and its subseciuent incorporation into DNA (Figure 1 0.261). Fluorine was chosen to replace hydrogen at DNA (Figure 1 0.261). Fluorine was chosen to replace hydrogen at the C5 position of uracil because it is of a similar size to hydrogen the C5 position of uracil because it is of a similar size to hydrogen (atomic radii: F. 0.13 nm: 1-1. 0.l2nrn). It was thought that this (atomic radii: F. 0.13 nm: 1-1. 0.l2nrn). It was thought that this similarity in size would give a drug that ould cause little steric similarity in size would give a drug that ould cause little steric disturbance to the biosynthetic pathway as well as being chemically disturbance to the biosynthetic pathway as well as being chemically inert. Analogues containing larger halogen atoms do not have any inert. Analogues containing larger halogen atoms do not have any appreciable activity.appreciable activity.
• FUdRP inhibits the enzyme thyniidylate synthetaseFUdRP inhibits the enzyme thyniidylate synthetase,, which in its which in its normal role is responsible for the transfer (if a meth\ I group normal role is responsible for the transfer (if a meth\ I group from the coenzyme from the coenzyme mmelhylenetetrahydrofolic acid (MeFI 14) to elhylenetetrahydrofolic acid (MeFI 14) to the C5 atom of deoxyuridylic acid (UdRP). The presence of the the C5 atom of deoxyuridylic acid (UdRP). The presence of the unreactive C5 F bond in FUdRP blocks this methylation, which unreactive C5 F bond in FUdRP blocks this methylation, which prevents the formation of deoxythymidylic acid (TdRP) and its prevents the formation of deoxythymidylic acid (TdRP) and its subseciuent incorporation into DNA (Figure 1 0.261). Fluorine subseciuent incorporation into DNA (Figure 1 0.261). Fluorine was chosen to replace hydrogen at the C5 position of uracil was chosen to replace hydrogen at the C5 position of uracil because it is of a similar size to hydrogen (atomic radii: F. 0.13 because it is of a similar size to hydrogen (atomic radii: F. 0.13 nm: 1-1. 0.l2nrn). It was thought that this similarity in size would nm: 1-1. 0.l2nrn). It was thought that this similarity in size would give a drug that ould cause little steric disturbance to the give a drug that ould cause little steric disturbance to the biosynthetic pathway as well as being chemically inert. biosynthetic pathway as well as being chemically inert. Analogues containing larger halogen atoms do not have any Analogues containing larger halogen atoms do not have any appreciable activity.appreciable activity.
prof. azaprof. aza
•The presence of the unreactive The presence of the unreactive C5 F bond in FUdRP blocks this C5 F bond in FUdRP blocks this methylation, which prevents the methylation, which prevents the formation of deoxythymidylic formation of deoxythymidylic acid (TdRP) and its subsecuent acid (TdRP) and its subsecuent incorporation into DNA (Figure 1 incorporation into DNA (Figure 1 0.261). 0.261).
prof. azaprof. aza
•Fluorine was chosen to Fluorine was chosen to replace hydrogen at the C5 replace hydrogen at the C5 position of uracil because it is position of uracil because it is of a similar size to hydrogen of a similar size to hydrogen (atomic radii: F. 0.13 nm: 1-1. (atomic radii: F. 0.13 nm: 1-1. 0.l2n0.l2nmm). ).
prof. azaprof. aza
• It was thought that this similarity in It was thought that this similarity in size would give a drug that could size would give a drug that could cause little steric disturbance to the cause little steric disturbance to the biosynthetic pathway as well as biosynthetic pathway as well as being chemically inert. Analogues being chemically inert. Analogues containing larger halogen atoms do containing larger halogen atoms do not have any appreciable activity.not have any appreciable activity.
prof. azaprof. aza
prof. azaprof. aza
Figure 10.26. (a) Examples of pyrimidines that act as antimetabolites. It should be noted that cytarabine only differs from cytidine by the stereochemistry of the 2’ carbon. (b) The intervention of fluorouracil in pyrimidine biosynthesis.
prof. azaprof. aza
Figure 10.27. Examples of topoisomerase inhibitors. Ellipticene acts h intercalation and inhibition of topoisomerase II enzymes. It is active against nasophar ngeal carcinomas. Amsacrinc is used to treat o arian carcinoflms. lvmphoinas and myelogenous leukaemias. (‘amptotheci n is an antituinour n1.
prof. azaprof. aza
13.213.2.. Enzyme Inhibitors Enzyme Inhibitors
• Enzyme inhibitors may he classified Enzyme inhibitors may he classified for convenience as those for convenience as those that inhibit that inhibit the enzymes directly responsible for the enzymes directly responsible for the formation of nucleic acids the formation of nucleic acids or the or the variety of variety of enzymes that catalyse the enzymes that catalyse the various stages in the formation of the various stages in the formation of the pirimidine pirimidine and purine bases required and purine bases required for the formation of nucleic acids.for the formation of nucleic acids.
prof. azaprof. aza
13.2.1 Topoisomerases13.2.1 Topoisomerases
• Topoisomerases are a group of Topoisomerases are a group of enzymes that are responsible enzymes that are responsible for the for the supercoiling, the cleavage and supercoiling, the cleavage and rejoining of DNA. rejoining of DNA.
• Their inhibition has the effect of Their inhibition has the effect of preventing transcriptionpreventing transcription. A number . A number of compounds (Figure 10.27) are of compounds (Figure 10.27) are believed to act by inhibiting these believed to act by inhibiting these enzymes. enzymes.
• It is thought that some It is thought that some intercalators act in this manner intercalators act in this manner although it is not clear whether although it is not clear whether the drug the drug bbinds to the inds to the topoisotopoisommerase prior to or after erase prior to or after the enzyme has formed a DNA—the enzyme has formed a DNA—enzyme complex.enzyme complex.
prof. azaprof. aza
prof. azaprof. aza
113.2.2 Enzyme Inhibitors for Purine 3.2.2 Enzyme Inhibitors for Purine and Primidine Precursor Systemsand Primidine Precursor Systems
• A wide range of compounds are active against a A wide range of compounds are active against a number of the enzyme s stems that arnumber of the enzyme s stems that are e involved in the involved in the biosynthesis of purines and pyrimidines in bacteria. biosynthesis of purines and pyrimidines in bacteria.
• In both of these examples the overall effect is the In both of these examples the overall effect is the inhibit ion of purine and pyrirnidine synthesis, which inhibit ion of purine and pyrirnidine synthesis, which results in theresults in the inhibition of the synthesis of DNA. This inhibition of the synthesis of DNA. This restricts the groth of the bacteria and ultimately pre restricts the groth of the bacteria and ultimately pre vents it from replicating, which gives the bodys natural vents it from replicating, which gives the bodys natural defences time to destroy the bacteria. Because defences time to destroy the bacteria. Because su]phonamides and trimethoprim inhibit different su]phonamides and trimethoprim inhibit different stages in the same metabolic pathway, they are often stages in the same metabolic pathway, they are often used in conjunction (Figure 1 0.28).This allows the used in conjunction (Figure 1 0.28).This allows the clinician to use lo ci and there fore safer doses.clinician to use lo ci and there fore safer doses.
• For exampleFor example, , sulphonamides inhibit sulphonamides inhibit dihydropteroate sdihydropteroate syynthetase (see nthetase (see section 6.12.1), which prevents the section 6.12.1), which prevents the formation of folic acid, whereas formation of folic acid, whereas trimethoprim inhibits dihydrofolate trimethoprim inhibits dihydrofolate reductase, which prevents the reductase, which prevents the conversion of folic acid to conversion of folic acid to tetrahydrofolate (see section tetrahydrofolate (see section 10.13.1.1).10.13.1.1).
prof. azaprof. aza
• In both of these examples the overall In both of these examples the overall effect is the effect is the inhibit ion of purine and inhibit ion of purine and pyripyrimimidine synthesisdine synthesis, which results in , which results in thethe inhibition of the synthesis of DNA. inhibition of the synthesis of DNA. This restricts the groThis restricts the growwth of the bacteria th of the bacteria and ultimately and ultimately prevents it from prevents it from replicating,replicating, which gives the bodys which gives the bodys natural defences time to destroy the natural defences time to destroy the bacteria. bacteria.
prof. azaprof. aza
• Because suBecause sullphonamides and phonamides and trimethoprim trimethoprim inhibit different stages inhibit different stages in the same metabolic pathwayin the same metabolic pathway, they , they are often used in conjunction (Figure are often used in conjunction (Figure 1 0.28).This allows the clinician to 1 0.28).This allows the clinician to use louse lowerwer and therefore safer doses. and therefore safer doses.
prof. azaprof. aza
prof. azaprof. aza
Figure 10.28. Sequential blocking using sulphamethoxazole and Trimethoprim.
prof. azaprof. aza
13.313.3.. Intercalating Agents Intercalating Agents
• Intercalating agents are compounds Intercalating agents are compounds that insert themselves between the that insert themselves between the bases of the DNA helix (Figure bases of the DNA helix (Figure 10.29). 10.29). This insertion causes the DNA This insertion causes the DNA helix to partially unwind at the site of helix to partially unwind at the site of the intercalated moleculethe intercalated molecule. .
• This inhibits transcription, which This inhibits transcription, which blocks the replication process of the blocks the replication process of the cell containing the DNA. cell containing the DNA.
• However, it is not known how the However, it is not known how the partial unwinding presents partial unwinding presents transcription transcription bbut some workers think ut some workers think that it inhibits topoisomerases (see that it inhibits topoisomerases (see section 10.12.2.1). Inhibition of cell section 10.12.2.1). Inhibition of cell replication can lead to cell death, replication can lead to cell death, which reduces the swhich reduces the sizeize of a tumour, of a tumour, the number of ‘free’ cancer cells or the number of ‘free’ cancer cells or the degree of infection, all of which the degree of infection, all of which will contribute to improving the health will contribute to improving the health of the patient.of the patient.
prof. azaprof. aza
prof. azaprof. aza
Figure 10.29. A schematic representation of the distortion of the DNA helix by intercalating agents. The horizontal lines represent the hydrogen-bonded bases. The rings of these bases and intercalating agent are edgeon to the reader.
prof. azaprof. aza
• The insertion of an intercalation The insertion of an intercalation agent appears to occur via either the agent appears to occur via either the minor or major grooves of DNA. minor or major grooves of DNA. Compounds that act as intercalating Compounds that act as intercalating agents must have structures that agents must have structures that contain a flat fused aromatic or contain a flat fused aromatic or heteroarornatic ring section that can heteroarornatic ring section that can fit between the flat structures of the fit between the flat structures of the bases of the DNA. bases of the DNA.
• It is believed that these aromatic It is believed that these aromatic structures are held in place by structures are held in place by hydrogen bonds, van der Waals’ hydrogen bonds, van der Waals’ forces and charge-transfer bonds forces and charge-transfer bonds (see section 5.2).(see section 5.2).
prof. azaprof. aza
prof. azaprof. aza
Figure 10.30. Examples of intercalating agents. Trade name.
prof. azaprof. aza
• Drugs whose mode of action includes Drugs whose mode of action includes intercalation arc the antimalarials intercalation arc the antimalarials quinine and chquinine and chlloroquine, the oroquine, the anticancer agents mitoxantrone and anticancer agents mitoxantrone and doxoruhicin, and the antibiotic doxoruhicin, and the antibiotic proflavine (Figure 10.30). proflavine (Figure 10.30).
• In each of these compounds it is the In each of these compounds it is the flat aromatic ring system that is flat aromatic ring system that is responsible for the intercalation. responsible for the intercalation.
• However, other groups in the However, other groups in the structures may also contribute to the structures may also contribute to the binding of a drug to the DNA. binding of a drug to the DNA.
• For example, the amino group of the For example, the amino group of the sugar residue of doxorubicin forms sugar residue of doxorubicin forms an ionic bond with the negatively an ionic bond with the negatively charged oxygens of the phosphate charged oxygens of the phosphate groups of the DNA chain, which groups of the DNA chain, which effectively locks the drug into place. effectively locks the drug into place.
prof. azaprof. aza
• A number of other drugs appear to A number of other drugs appear to have groups that act in a similar have groups that act in a similar manner.manner.
• Some intercalating agents xhibit a Some intercalating agents xhibit a preference br certain combinations of preference br certain combinations of bases in DNA. For example, bases in DNA. For example, mitoxantrone appears to prefer to mitoxantrone appears to prefer to intercalate with cytosine—guanosine-intercalate with cytosine—guanosine-rich sequences. This type of behaviour rich sequences. This type of behaviour does open out the possibility of does open out the possibility of selective action in some cases.selective action in some cases.
prof. azaprof. aza
prof. azaprof. aza
13.4 Alkylating Agents13.4 Alkylating Agents
• Alkylating agents are believed to bond to Alkylating agents are believed to bond to the nucleic acid chains in either the the nucleic acid chains in either the major or minor grooves. In DNA the major or minor grooves. In DNA the alkylating agent frequently forms either alkylating agent frequently forms either intrastrand or inlerstrand crosslinks. intrastrand or inlerstrand crosslinks. Intrastrand cross-linking agents form a Intrastrand cross-linking agents form a bridge between two parts of the same bridge between two parts of the same chain (Figure 10.31). This has the elfect chain (Figure 10.31). This has the elfect of distorting the strand, which inhibits of distorting the strand, which inhibits transcription.transcription.
prof. azaprof. aza
Figure 10.31. A schematic representation of the intrastrand cross-linking.
prof. azaprof. aza
• lnterstrand cross—links are formed lnterstrand cross—links are formed between the two separate chains of between the two separate chains of the DNAthe DNA,which,which has the has the effect of effect of locking them together (Figure 10.32). locking them together (Figure 10.32). This also inhibits transcription. This also inhibits transcription.
• In RNA only intrastrand cross-links In RNA only intrastrand cross-links are possible. However, irrespective of are possible. However, irrespective of whether or not it forms a bridge.whether or not it forms a bridge. the the bonding of an alkylating agent to a bonding of an alkylating agent to a nucleic acid inhibits replication of nucleic acid inhibits replication of that nucleic acidthat nucleic acid
• InIn the case of bacteria this prevents the case of bacteria this prevents an increase in the sian increase in the sizeze of the of the infection and so buys the bod’infection and so buys the bod’ time time for its immune system to destroy the for its immune system to destroy the existing bacteria. However, in the existing bacteria. However, in the case of cancer itcase of cancer it may lead to cell may lead to cell death and a benefdeath and a benefiicial reduction in cial reduction in tumour stumour sizeize..
prof. azaprof. aza
prof. azaprof. aza
Figure 10.32. (a) The general structure of nitrogen mustards (h) The proposee mechanism for tormimu’ interstrandcross-links by the action of aliphatic nitrogen mustards.
prof. azaprof. aza
• The nucleophilic nature of the nucleic The nucleophilic nature of the nucleic acids means that alkylating agents acids means that alkylating agents are usuallyare usually electrophiles or give rise electrophiles or give rise to electrophiles. to electrophiles.
• For example, it is believed that a For example, it is believed that a weaklyweakly electrophilic β-carbon atom of electrophilic β-carbon atom of an aliphatic nitrogen mustard an aliphatic nitrogen mustard alkylating agent, such asalkylating agent, such as mechlorethamine (Mustine), mechlorethamine (Mustine),
• is converted to the more highly is converted to the more highly electrophilic aziridine ion by an internal electrophilic aziridine ion by an internal nucleophilic substitution of a b-chlorine nucleophilic substitution of a b-chlorine atom. This is thought to be followedatom. This is thought to be followed
• by the nucleophilic attack of the N7 of by the nucleophilic attack of the N7 of a guanine residue on this ion by what a guanine residue on this ion by what appears to bean SN2 type of appears to bean SN2 type of mechanism. mechanism.
prof. azaprof. aza
• Since these drugs have two Since these drugs have two hydrocarbon chains with b-hydrocarbon chains with b-chlorogroups, each of these chlorogroups, each of these chlorogroups is believed to react with chlorogroups is believed to react with a guanine residue in aa guanine residue in a different chain different chain of the DNA strand to form a cross-of the DNA strand to form a cross-link between the two nucleic acidlink between the two nucleic acid chains (Fig. 10.38).chains (Fig. 10.38).
prof. azaprof. aza
prof. azaprof. aza
Figure 10.33. (a) The structure of chiorambucil and (h) a proposed mode of action for some aromatic nitrogen mustards.
prof. azaprof. aza
• The electrophilic nature of alkylating The electrophilic nature of alkylating agents means that they can also agents means that they can also react with a wide variety of other react with a wide variety of other nucleophilic biomaromolecules. nucleophilic biomaromolecules.
• This accounts for many of the unwanted This accounts for many of the unwanted toxic effects that are frequently toxic effects that are frequently observed with the use of these drugs. In observed with the use of these drugs. In the case of the nitrogen mustards. the case of the nitrogen mustards. attempts to reduce these side effects attempts to reduce these side effects have centred on reducing their reactivity have centred on reducing their reactivity by discouraging the formation of the by discouraging the formation of the aiiridine ion before the drug reaches its aiiridine ion before the drug reaches its site of action. site of action.
prof. azaprof. aza
• The approach adopted has been to The approach adopted has been to reduce the nucleophilic character of reduce the nucleophilic character of the nitrogen atom by attaching ii to the nitrogen atom by attaching ii to an electron-withdrawing aromatic an electron-withdrawing aromatic ring. This produced analogues that ring. This produced analogues that would only react with strong would only react with strong nucleophiles and resuited in the nucleophiles and resuited in the development of chlorambucil. development of chlorambucil.
prof. azaprof. aza
• This drug is one of the least toxic This drug is one of the least toxic nitrogen mustards, being active nitrogen mustards, being active against malignant lymphomasagainst malignant lymphomas,, carcinomas of the breast and ovary carcinomas of the breast and ovary and lymphocytic and lymphocytic lleukaemia. eukaemia.
prof. azaprof. aza
• It has been suggested that because It has been suggested that because of the reduction in the nucleophilicity of the reduction in the nucleophilicity of the nitrogen atom these aromatic of the nitrogen atom these aromatic nitrogen mustards do not form an nitrogen mustards do not form an aziriaziriddine ion. Instead they react by ine ion. Instead they react by direct substitution of the 13-chlorine direct substitution of the 13-chlorine atoms by guanineatoms by guanine,, which is a strong which is a strong nucleophile, by an S.I type of nucleophile, by an S.I type of mechanism (Figure 10.33).mechanism (Figure 10.33).
prof. azaprof. aza
prof. azaprof. aza
Figure 10.34. Cvclophospliamide and the formation ol phosphorainide mustnrd. the iictl\c hum sit this di ui.
prof. azaprof. aza
• Further attempts to reduce the Further attempts to reduce the toxicity otoxicity off nitrogen mustards were nitrogen mustards were based on making the drug more based on making the drug more selective. o approaches have yielded selective. o approaches have yielded useful drugs. The first was based on useful drugs. The first was based on the fact that the rapid synthesis of the fact that the rapid synthesis of proteins that occurs in tumour cells proteins that occurs in tumour cells requires a large supply of amino acid requires a large supply of amino acid raw material from outside the cell. raw material from outside the cell.
• Consequently. it was thought that Consequently. it was thought that the presence of an anuno acid the presence of an anuno acid residue in the structure of a nitrogen residue in the structure of a nitrogen mustard might lead to an increased mustard might lead to an increased uptake of that compound. This uptake of that compound. This approach resulted in the synthesis of approach resulted in the synthesis of the phenvlalanine mustard the phenvlalanine mustard meiphalan (iible I t).6meiphalan (iible I t).6
prof. azaprof. aza
• The —form of this drug is more The —form of this drug is more active than the 1)—form and so it active than the 1)—form and so it has been sughas been sug
• gested that the L—form may be gested that the L—form may be transported into the cell by means of transported into the cell by means of an i -phen lalanine activean i -phen lalanine active
• transport system.transport system.
prof. azaprof. aza
prof. azaprof. aza
• The second approach was based on the fact that some tumours \ere thought to The second approach was based on the fact that some tumours \ere thought to contain a highcontain a high
• concentration of phosphoramidases. This resulted in the synthesis of nitrogen concentration of phosphoramidases. This resulted in the synthesis of nitrogen mustard anamustard ana
• mechanism. logues whose structures contained phosphorus functional groups that mechanism. logues whose structures contained phosphorus functional groups that could he attacked bycould he attacked by
• this enzyme. It led to the development of the cyclophosphamide (Figure 10.34). this enzyme. It led to the development of the cyclophosphamide (Figure 10.34). which haswhich has
• a wide spectrum of activity. However, the action of this prodrug has now been shown a wide spectrum of activity. However, the action of this prodrug has now been shown toto
• he due to phosphoramide mustard formed by oxidation by microsomal enzymes in he due to phosphoramide mustard formed by oxidation by microsomal enzymes in the liverthe liver
• rather than hydrolysis by tumour phosphoramidases. The acrolein produced in this rather than hydrolysis by tumour phosphoramidases. The acrolein produced in this proceproce
• is he-lieved to he the source of mvelosuppression and haemorrhagic cvstitis is he-lieved to he the source of mvelosuppression and haemorrhagic cvstitis associatedassociated
• with the use of cvclophosphamide. However, co—administration of the drug with with the use of cvclophosphamide. However, co—administration of the drug with sodiumsodium
• 2-mercaptoethanesulphonate (MESNA) can relieve some of these symptoms. MESNA 2-mercaptoethanesulphonate (MESNA) can relieve some of these symptoms. MESNA formsforms
• a water-soluble adduct with the acrolein, which is then excreted in the urine.a water-soluble adduct with the acrolein, which is then excreted in the urine.
prof. azaprof. aza
• Some alkylating agents act by decomposing to Some alkylating agents act by decomposing to produce an electrophile that bonds to a produce an electrophile that bonds to a nudeiophilic group of a base in the nucleic acid. For nudeiophilic group of a base in the nucleic acid. For example. temozolomide (Table 10.() entersthe example. temozolomide (Table 10.() entersthe major groove of DNA where it reacts with water to major groove of DNA where it reacts with water to from nitrogen. carbon dioxide, an aniniomdazole from nitrogen. carbon dioxide, an aniniomdazole and a methyl carbonium ion (CH3). This methyl and a methyl carbonium ion (CH3). This methyl carhonium ion then methlates the strongly carhonium ion then methlates the strongly nucleophilic N7 of the guanine bases in the major nucleophilic N7 of the guanine bases in the major groo e. A range of different classes of compound groo e. A range of different classes of compound can act as nucleic acid alkylating agents lable can act as nucleic acid alkylating agents lable 10.6). Within these classes a ii umber of 10.6). Within these classes a ii umber of compounds have been fL1I1d to he usc i ul di ugs. compounds have been fL1I1d to he usc i ul di ugs. Iii mam’, cases their effectiveness is improved by Iii mam’, cases their effectiveness is improved by the use of combinations of drugs. Their modes of the use of combinations of drugs. Their modes of action are usually not fully understood but a large action are usually not fully understood but a large amount of information is available concerning their amount of information is available concerning their structure-action relationships.structure-action relationships.
prof. azaprof. aza
Table 10.6. Some examples of the classes and compounds of anticancer agents that act by aik atioti ol nucleic acids. 11 is emphasised that this table only lists some of the classes of alkylating compound that are active against cancers.
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prof. azaprof. aza
Figure 10.35. Development routes for antisense drugs. Examples of: (a) a section of the backbone of a deoxy ribonuCWICIId cleic chain; (b) backbone modifications; (c) sugar residue modifications; and (d) base modifications,
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10.13.5 Antisense Drugs10.13.5 Antisense Drugs
• The concept of antisense compounds or sequence-defined The concept of antisense compounds or sequence-defined oligonucleotides (ONs) offers a newoligonucleotides (ONs) offers a new
• specific approach to designing drugs that target nucleic specific approach to designing drugs that target nucleic acids, The idea underlying this approachacids, The idea underlying this approach
• is that the antisense compound contains the sequence of is that the antisense compound contains the sequence of complementary bases to those foundcomplementary bases to those found
• in a short section of the target nucleic acid. This section is in a short section of the target nucleic acid. This section is usually part of the genetic message being carried by an usually part of the genetic message being carried by an mRNA molecule. The antisensc compound binds to this mRNA molecule. The antisensc compound binds to this section by hvdrogen bonding between the complementary section by hvdrogen bonding between the complementary base pairs. This inhibits translation of the message carried base pairs. This inhibits translation of the message carried by the mRNA, which inhibits the production of a specific by the mRNA, which inhibits the production of a specific protein responsible for a disease state in a patient.protein responsible for a disease state in a patient.
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• Antisense compounds were originally short Antisense compounds were originally short lengths of nucleic acid chains that had lengths of nucleic acid chains that had base sequences that were complementary base sequences that were complementary to those found in their target RNA. These to those found in their target RNA. These short lengths of nucleic acid antisense short lengths of nucleic acid antisense compounds were found to be unsuitable as compounds were found to be unsuitable as drugs because of poor binding to the drugs because of poor binding to the target site and short half-lives due to target site and short half-lives due to enzyme action. However, they provided enzyme action. However, they provided lead compounds for further des elopment lead compounds for further des elopment (Figure 10.35). Development is currently (Figure 10.35). Development is currently taking three basic routes:taking three basic routes:
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flg.re fl The bleomycin’. The drug bleomycin sulphate is a mixture of a number of bleomycins.
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• (I) modification of the backbone linking the bases to (I) modification of the backbone linking the bases to increase resistance to ene’ymic hydro sis:increase resistance to ene’ymic hydro sis:
• (ii) changing the nature of the sugar residue by (ii) changing the nature of the sugar residue by either replacing some of the free Ii) droxy groups either replacing some of the free Ii) droxy groups by other substituents or forming derivatives of by other substituents or forming derivatives of these groupcthese groupc
• (iii) modifying the nature of the substituent groups (iii) modifying the nature of the substituent groups of the bases.of the bases.
• Antisense compounds are able to bind to both RNA Antisense compounds are able to bind to both RNA and DNA. In the latter case they form a triple helix. and DNA. In the latter case they form a triple helix. At present, antisense drugs are still in the early At present, antisense drugs are still in the early stages of their development but the concept has stages of their development but the concept has aroused considerable interest in the aroused considerable interest in the pharmaceutical industrpharmaceutical industr
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10.13.6 Chain-c(eavlng 10.13.6 Chain-c(eavlng AgentsAgents• The interaction ot chain-cleiving agents with DNA The interaction ot chain-cleiving agents with DNA
results in the breaking of the nucleic aciJ into results in the breaking of the nucleic aciJ into fragments. Currently, the main cleaving agents are fragments. Currently, the main cleaving agents are the bleomycins (Figure 10.36) and their analogues. the bleomycins (Figure 10.36) and their analogues. However, other classes of drug are in the However, other classes of drug are in the development stage.development stage.
• The bleomycins are a group of naturally occurring The bleomycins are a group of naturally occurring glycoproteins that exhibit antitumour activity. glycoproteins that exhibit antitumour activity. When administered to patients they tend to When administered to patients they tend to accumulate in the squamous cells and so are useful accumulate in the squamous cells and so are useful for treating cancers of the head, neck and genitalia. for treating cancers of the head, neck and genitalia. However, the bleomycins cause pain and ulceration However, the bleomycins cause pain and ulceration of areas of skin that contain a high concentration of of areas of skin that contain a high concentration of keratin, as well as other unwanted side effects.keratin, as well as other unwanted side effects.
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• The action of the bleomycins is not fully The action of the bleomycins is not fully understood. It is believed that the bithiarole moiet understood. It is believed that the bithiarole moiet (domain X in Figure 10.36) intercalates with the (domain X in Figure 10.36) intercalates with the DNA. In bleomycin A3 the resulting adductto the DNA. In bleomycin A3 the resulting adductto the receptor of the host cell the virus—receptor receptor of the host cell the virus—receptor complex is transported into the cell by receptor-complex is transported into the cell by receptor-mediated endocytosis (see section 4.3.6). In the mediated endocytosis (see section 4.3.6). In the course of this process the protein capsid and any course of this process the protein capsid and any lipoprotein envelopes may be removed. Once it has lipoprotein envelopes may be removed. Once it has entered the host cell the viral nucleic acid is able entered the host cell the viral nucleic acid is able to use the host’s cellular machinery to synthesise to use the host’s cellular machinery to synthesise the nucleic acids and proteins required to produce the nucleic acids and proteins required to produce a number ofnew viruses (Figure 10.38).a number ofnew viruses (Figure 10.38).
14. Viruses14. Viruses• Viruses are infective agents that are Viruses are infective agents that are
considerably smaller than bacteria. considerably smaller than bacteria. They are essentiallyThey are essentially packages, packages, known as virions, of chemicals that known as virions, of chemicals that invade host cells. invade host cells.
• However, viruses are notHowever, viruses are not independent and can only penetrate independent and can only penetrate a host cell that can a host cell that can satisfy the satisfy the specific needs specific needs of thatof that virus. virus.
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• The mode of penetration varies The mode of penetration varies considerably from virus to virus. Once considerably from virus to virus. Once inside the hostinside the host cell viruses take over cell viruses take over the metabolic machinery of the host the metabolic machinery of the host and use it to produce moreand use it to produce more viruses. viruses. Replication is often lethal to the host Replication is often lethal to the host cell, which may undergo lysis to cell, which may undergo lysis to release therelease the progeny of the virus.progeny of the virus.
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•However, in some cases the virus However, in some cases the virus may integrate into the hostmay integrate into the host chromosome chromosome and become and become dormant. The ability of viruses to dormant. The ability of viruses to reproduce means that they canreproduce means that they can be be regarded regarded as being on the as being on the borderline of being living borderline of being living organismsorganisms..
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14.1. Structure and 14.1. Structure and replicationreplication
•Viruses consist of a core of either Viruses consist of a core of either DNA or, as in the majority of cases, DNA or, as in the majority of cases, RNA fully orRNA fully or partially covered by a partially covered by a protein coating known as the protein coating known as the capsid. The capsid consists of a capsid. The capsid consists of a numbernumber of polypeptide molecules of polypeptide molecules known as capsomers (Fig.10.43).known as capsomers (Fig.10.43).
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prof. azaprof. aza
Figure 10.37. (a) Schematic representations of the structure of a virus (a) without a lipoprotein envelope (naked virus) and (h) with a lipoprotein envelope.
• The capsid that surrounds mostThe capsid that surrounds most viruses viruses consists of a number of different consists of a number of different capsomers although some viruses will capsomers although some viruses will havehave capsids that only contain one capsids that only contain one type of capsomer. It is the type of capsomer. It is the arrangement of the capsomersarrangement of the capsomers around around the nucleic acid that determines the the nucleic acid that determines the overall shape of the virion.overall shape of the virion.
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• In the majority ofIn the majority of viruses, the viruses, the capsomers form a layer or capsomers form a layer or several layers that completely several layers that completely surround the nucleicsurround the nucleic acids. acids. However, there are some viruses However, there are some viruses in which the capsomers form an in which the capsomers form an open-ended tubeopen-ended tube that holds the that holds the nucleic acids.nucleic acids.
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• In many viruses the capsid is coated In many viruses the capsid is coated with a protein-containing lipid bilayer with a protein-containing lipid bilayer membrane.membrane. These are known as These are known as enveloped viruses. Their lipid bilayers enveloped viruses. Their lipid bilayers are often derived from theare often derived from the plasma plasma membrane of the host cell and are membrane of the host cell and are formed when the virus leaves the host formed when the virus leaves the host cell by acell by a process known as budding.process known as budding.
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• Budding is a mechanism by which a Budding is a mechanism by which a virus leaves a host cellvirus leaves a host cell without killing without killing that cell. that cell. It provides the virus with a It provides the virus with a membrane whose lipid components membrane whose lipid components areare identical to those of the host (Fig. identical to those of the host (Fig. 10.43). This 10.43). This allows the virus to allows the virus to penetrate new host cellspenetrate new host cells without without activating the host’s, immune activating the host’s, immune systems.systems.
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• Viruses bind to host cells at specific Viruses bind to host cells at specific receptor sites on the host’s cell receptor sites on the host’s cell envelope.envelope. The binding sites on the The binding sites on the virus are polypeptides in its capsid or virus are polypeptides in its capsid or lipoprotein envelope.lipoprotein envelope. Once the virus Once the virus has bound to the receptor of the host has bound to the receptor of the host cell the virus–receptor complexcell the virus–receptor complex is is transported into the cell by receptor-transported into the cell by receptor-mediated endocytosis.mediated endocytosis.
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• InIn the course of this process the the course of this process the protein capsid and any lipoprotein protein capsid and any lipoprotein envelopes may beenvelopes may be removed. Once it removed. Once it has entered the host cell the viral has entered the host cell the viral nucleic acid is able to use the host’snucleic acid is able to use the host’s cellular machinery to synthesise the cellular machinery to synthesise the nucleic acids and proteins required to nucleic acids and proteins required to replicatereplicate a number of new viruses (Fig. a number of new viruses (Fig. 10.44).10.44).
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•A great deal of information is A great deal of information is availableavailable concerning the details of concerning the details of the mechanism of virus replication the mechanism of virus replication but this text will onlybut this text will only outline the outline the main points. For greater detail the main points. For greater detail the reader is referred to specialist reader is referred to specialist texts ontexts on virology.virology.
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14.2. Classification14.2. Classification
• RNA-viruses can be broadly classified RNA-viruses can be broadly classified into two general types, namely: RNA-into two general types, namely: RNA-viruses andviruses and RNA-retroviruses.RNA-retroviruses.
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• Figure 10.44 A schematic representation of the replication of Figure 10.44 A schematic representation of the replication of RNA-virusesRNA-viruses
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RNA-virusesRNA-viruses
•RNA-virus replication usually RNA-virus replication usually occurs entirely in the occurs entirely in the cytoplasm. The viral mRNA cytoplasm. The viral mRNA eithereither forms part of the RNA forms part of the RNA carried by the virion or is carried by the virion or is synthesised by an enzyme synthesised by an enzyme already presentalready present in the virion. in the virion.
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•This viral mRNA is used to This viral mRNA is used to produce the necessary viral produce the necessary viral proteins by translation using proteins by translation using the host cell’s ribosomes and the host cell’s ribosomes and enzyme systems.enzyme systems.
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•Some of the viral proteins areSome of the viral proteins are enzymes that are used to catalyse enzymes that are used to catalyse the reproduction of more viral the reproduction of more viral mRNA. The new viralmRNA. The new viral RNA and RNA and viral proteins are assembled into viral proteins are assembled into a number of new virions that are a number of new virions that are ultimatelyultimately released from the host released from the host cell by either lysis or buddingcell by either lysis or budding..
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RetrovirusesRetroviruses• Retroviruses Retroviruses synthesise viral DNA synthesise viral DNA
using their viral RNA as a using their viral RNA as a templatetemplate. .
• This process isThis process is catalysed by enzyme catalysed by enzyme systems known as systems known as reverse reverse transcriptasestranscriptases that form part of the that form part of the virion. Thevirion. The viral DNA is incorporated viral DNA is incorporated into the host genome to form a so-into the host genome to form a so-called called provirus.provirus.
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•Transcription ofTranscription of the provirus the provirus produces new ‘genomic’ viral produces new ‘genomic’ viral RNA and viral mRNA. The viral RNA and viral mRNA. The viral mRNA is used tomRNA is used to produce viral produce viral proteins, which together with the proteins, which together with the ‘genomic’ viral RNA are ‘genomic’ viral RNA are assembled into newassembled into new virions. virions.
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•These virions are released by These virions are released by budding , which in many budding , which in many cases does not kill the host cases does not kill the host cell. Retroviruses are cell. Retroviruses are responsible for some forms of responsible for some forms of cancer and AIDScancer and AIDS
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DNA-virusesDNA-viruses
• Most DNA-viruses enter the host cell’s Most DNA-viruses enter the host cell’s nucleus where formation of viral mRNA nucleus where formation of viral mRNA byby transcription from the viral DNA is transcription from the viral DNA is brought about by the host cell’s brought about by the host cell’s polymerases. This viralpolymerases. This viral mRNA is used mRNA is used to produce viral proteins by translation to produce viral proteins by translation using the host cell’s ribosomes andusing the host cell’s ribosomes and enzyme systems.enzyme systems.
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• Some of these proteins will be Some of these proteins will be enzymes that can catalyse the enzymes that can catalyse the synthesis ofsynthesis of more viral DNA. more viral DNA.
• This DNA and the viral proteins This DNA and the viral proteins synthesised in the host cell are synthesised in the host cell are assembledassembled into a number of new into a number of new virions that are ultimately released virions that are ultimately released from the host by either cell lysisfrom the host by either cell lysis or or buddingbudding
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14.3. Viral diseases14.3. Viral diseases
• Viral infection of host cells is a common Viral infection of host cells is a common occurrence. Most of the time this occurrence. Most of the time this infection doesinfection does not result in illness as not result in illness as the body’s immune system can usually the body’s immune system can usually deal with such viral invasiondeal with such viral invasion..
• When illness occurs it is often short When illness occurs it is often short lived and leads to long-term immunity.lived and leads to long-term immunity.
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• However, aHowever, a number of viral infections can lead to serious number of viral infections can lead to serious medical conditions (. Somemedical conditions (. Some viruses like HIV, the aetiological viruses like HIV, the aetiological agent of AIDS, are able to remain dormant in the host foragent of AIDS, are able to remain dormant in the host for a a number of years before becoming active, whilst others such as number of years before becoming active, whilst others such as herpes zoster (shingles)herpes zoster (shingles) can give rise to recurrent bouts of the can give rise to recurrent bouts of the illness. Both chemotherapy and preventativeillness. Both chemotherapy and preventative
• vaccination are used to treat vaccination are used to treat patients. The latter is the main patients. The latter is the main clinical approach since it hasclinical approach since it has
• been difficult to design drugs that only target the virus. been difficult to design drugs that only target the virus. However, a number of antiviralHowever, a number of antiviral
• drugs have been developed and are in clinical use.drugs have been developed and are in clinical use.
• AIDSAIDS
• AIDSAIDS
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• Both chemotherapy and preventativeBoth chemotherapy and preventative vaccination are used to treat vaccination are used to treat patients. The latter is the main patients. The latter is the main clinical approach since it hasclinical approach since it has been been difficult to design drugs that only difficult to design drugs that only target the virus. However, a number target the virus. However, a number of antiviralof antiviral drugs have been drugs have been developed and are in clinical use.developed and are in clinical use.
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prof. azaprof. aza
AIDSAIDS
• AIDS is a disease that progressively AIDS is a disease that progressively destroys the human immune system. destroys the human immune system. It is caused by theIt is caused by the human human immunodeficiency virus (HIV), which immunodeficiency virus (HIV), which is a retrovirus. This virus is a retrovirus. This virus enters and enters and destroysdestroys human T4 lymphocyte cellshuman T4 lymphocyte cells. . These cells are a vital part of the These cells are a vital part of the human immune system.human immune system.
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•Their destruction reduces the Their destruction reduces the body’s resistance to other body’s resistance to other infectious diseases, such asinfectious diseases, such as pneumonia, and some rare forms pneumonia, and some rare forms of cancer.of cancer.
•..
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• The entry of the virus into the body The entry of the virus into the body usually causes an initial period of usually causes an initial period of acute ill health with the patient acute ill health with the patient suffering from headaches, fevers and suffering from headaches, fevers and rashes, amongst other symptoms.rashes, amongst other symptoms.
• This is followed by a period of This is followed by a period of relatively good healthy where the relatively good healthy where the virus replicates in the lymph nodes.virus replicates in the lymph nodes.
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• This relatively healthy period This relatively healthy period normally lasts a number of years normally lasts a number of years before fullblownbefore fullblown
• AIDS appears. Full-blown AIDS is AIDS appears. Full-blown AIDS is characterised by a wide variety of characterised by a wide variety of diseases suchdiseases such as bacterial infections, as bacterial infections, neurological diseases and cancers. neurological diseases and cancers. Treatment is more effective whenTreatment is more effective when a a mixture of antiviral agents is usedmixture of antiviral agents is used
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14.4. Antiviral drugs14.4. Antiviral drugs
• It has been found that viruses utilise It has been found that viruses utilise a number of virus-specific enzymes a number of virus-specific enzymes during replication.during replication.
• These enzymes and the processes These enzymes and the processes they control are significantly different they control are significantly different from those of thefrom those of the host cell to make host cell to make them a useful target for medicinal them a useful target for medicinal chemists.chemists.
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•Consequently, antiviral drugsConsequently, antiviral drugs normally act by inhibiting viral normally act by inhibiting viral nucleic acid synthesis, inhibiting nucleic acid synthesis, inhibiting attachment to andattachment to and penetration of penetration of the host cell or inhibiting viral the host cell or inhibiting viral protein synthesis.protein synthesis.
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Nucleic acid synthesis Nucleic acid synthesis inhibitorsinhibitors
• Nucleic acid synthesis inhibitors usually Nucleic acid synthesis inhibitors usually act by inhibiting the polymerases or act by inhibiting the polymerases or reversereverse transcriptases required for transcriptases required for nucleic acid chain formation. However, nucleic acid chain formation. However, because they are usuallybecause they are usually analogues of analogues of the purine and pyrimidine bases found in the purine and pyrimidine bases found in the viral nucleic acids, they are oftenthe viral nucleic acids, they are often incorporated into the growing nucleic incorporated into the growing nucleic acid chain.acid chain.
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• In this case their general mode of In this case their general mode of actionaction frequently involves conversion frequently involves conversion to the corresponding 50-triphosphate to the corresponding 50-triphosphate by the host cell’sby the host cell’s cellular kinases. cellular kinases. This conversion may also involve This conversion may also involve specific viral enzymes in the initialspecific viral enzymes in the initial monophosphorylation step.monophosphorylation step.
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• These triphosphate drug derivatives are These triphosphate drug derivatives are incorporated into theincorporated into the nucleic acid chain nucleic acid chain where they terminate its formation. where they terminate its formation. Termination occurs because the drugTermination occurs because the drug residues do not have the 30-hydroxy group residues do not have the 30-hydroxy group necessary for the phosphate ester formationnecessary for the phosphate ester formation required for further growth of the nucleic acid required for further growth of the nucleic acid chain. This effectively inhibits thechain. This effectively inhibits the polymerases and ranscriptases that catalyse polymerases and ranscriptases that catalyse the growth of the nucleic acid (Fig. 10.45the growth of the nucleic acid (Fig. 10.45).).
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•This effectively inhibits the This effectively inhibits the polymerases and ranscriptases polymerases and ranscriptases that catalyse the growth of the that catalyse the growth of the nucleic acid (Fig. 10.45).nucleic acid (Fig. 10.45).
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prof. azaprof. aza
AciclovirAciclovir• Aciclovir was the first effective Aciclovir was the first effective
antiviral drug. It is effective against a antiviral drug. It is effective against a number ofnumber of herpes viruses, notably herpes viruses, notably simplex, varicella-zoster (shingles), simplex, varicella-zoster (shingles), varicella (chickenpox) andvaricella (chickenpox) and Epstein–Epstein–Barr virus (glandular fever). It may Barr virus (glandular fever). It may be administered orally and by be administered orally and by intravenousintravenous injection as well as injection as well as topically. Orally administered doses topically. Orally administered doses have a low bioavailability.have a low bioavailability.
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• The action of aciclovir is more The action of aciclovir is more effective in virus-infected host cells effective in virus-infected host cells because the viralbecause the viral thymidine kinase is thymidine kinase is a more efficient catalyst for the a more efficient catalyst for the monophosphorylation of aciclovir monophosphorylation of aciclovir thanthan the thymidine kinases of the the thymidine kinases of the host cell.host cell.
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• This leads to an increase in the This leads to an increase in the concentration of theconcentration of the aciclovir aciclovir triphosphate, triphosphate, which has 100-fold which has 100-fold greater affinity for viral DNA greater affinity for viral DNA polymerase thanpolymerase than human DNA human DNA polymerase. polymerase.
• As a result, it preferentially As a result, it preferentially competitively inhibits viral DNAcompetitively inhibits viral DNA polymerase and so prevents the virus polymerase and so prevents the virus from replicating.from replicating.
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• However, resistance has beenHowever, resistance has been reported due to changes in the viral reported due to changes in the viral mRNA responsible for the production mRNA responsible for the production of the viralof the viral thymidine kinase. thymidine kinase. Aciclovir also acts by terminating Aciclovir also acts by terminating chain formation. chain formation. The aciclovir–DNAThe aciclovir–DNA complex complex formed by the drug also formed by the drug also irreversibly inhibits DNA polymerase.irreversibly inhibits DNA polymerase.
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VidarabineVidarabine
• Vidarabine is active against herpes Vidarabine is active against herpes simplex and herpes varicella-zoster.simplex and herpes varicella-zoster.
• However, the drug does give rise to However, the drug does give rise to nausea, vomiting, tremors, dizziness nausea, vomiting, tremors, dizziness and seizures. Inand seizures. In addition it has been addition it has been reported to be mutagenic, reported to be mutagenic, teratogenic and carcinogenic in teratogenic and carcinogenic in animal studiesanimal studies..
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• Vidarabine is administered by Vidarabine is administered by intravenous infusion and topical intravenous infusion and topical application. It has a half-lifeapplication. It has a half-life of about of about one hour, the drug being rapidly one hour, the drug being rapidly deaminated to arabinofuranosyl deaminated to arabinofuranosyl hypoxanthine (ara-HX) by adenosine hypoxanthine (ara-HX) by adenosine deaminase.deaminase.
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• This enzyme is found in the serum and red This enzyme is found in the serum and red blood cells. Ara-HX, which also exhibits a blood cells. Ara-HX, which also exhibits a weak antiviral action, has a half-life of weak antiviral action, has a half-life of about 3.5 hours.about 3.5 hours.
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prof. azaprof. aza
Zidovudine (AZT)Zidovudine (AZT)• Zidovudine was originally Zidovudine was originally
synthesised in 1964 as an analogue synthesised in 1964 as an analogue ofof thymine by J. Horwitz as a thymine by J. Horwitz as a potential antileukaemia drug. It was potential antileukaemia drug. It was found to be unsuitable for usefound to be unsuitable for use in this in this role and for 20 years was ignored, role and for 20 years was ignored, even though in 1974even though in 1974 W. Osterag et W. Osterag et al. al. reported thatreported that it was active against it was active against Friend leukaemia virusFriend leukaemia virus, a retrovirus., a retrovirus.
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•However, the identification in However, the identification in 19831983 of the retrovirus HIVas the of the retrovirus HIVas the source of AIDS resulted in the source of AIDS resulted in the virologist M. St Clair setting up avirologist M. St Clair setting up a screening programme for drugs screening programme for drugs that could attack HIVthat could attack HIV
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• Fourteen compounds were selected Fourteen compounds were selected and screened against Friend and screened against Friend leukaemia virus and a second leukaemia virus and a second retrovirus called Harvey sarcoma retrovirus called Harvey sarcoma virus. virus. This screen led to the This screen led to the discovery of zidovudine (AZT), which discovery of zidovudine (AZT), which was rapidly developed into clinical was rapidly developed into clinical use on selected patients in 1986use on selected patients in 1986..
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prof. azaprof. aza
•AZT is converted by the action of AZT is converted by the action of cellular thymidine kinase to the cellular thymidine kinase to the 50-triphosphate. This50-triphosphate. This inhibits the inhibits the enzyme reverse transcriptase in enzyme reverse transcriptase in the retrovirus, which effectively the retrovirus, which effectively prevents itprevents it from forming the viral from forming the viral DNA necessary for viral replicationDNA necessary for viral replication..
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• The incorporation of AZT intoThe incorporation of AZT into the the nucleic acid chain also results in nucleic acid chain also results in chain termination because the chain termination because the presence of the 30-azidepresence of the 30-azide group group prevents the reaction of the chain prevents the reaction of the chain with the 50-triphosphate of the next with the 50-triphosphate of the next nucleotidenucleotide waiting to join the chain waiting to join the chain (Fig. 10.45).(Fig. 10.45).
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•AZT is also active against AZT is also active against mammalian DNAmammalian DNA polymerase and polymerase and although its affinity for this although its affinity for this enzyme is about 100-fold less enzyme is about 100-fold less this action isthis action is thought to be the thought to be the cause of some of its unwanted cause of some of its unwanted side effects.side effects.
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• Zidovudine is active against the Zidovudine is active against the retroviruses (see section 10.14.2) retroviruses (see section 10.14.2) that cause AIDSthat cause AIDS (HIV virus) and (HIV virus) and certain types of leukaemia. certain types of leukaemia.
• It also inhibits cellular a-DNA It also inhibits cellular a-DNA polymerase butpolymerase but only at only at cconcentrations in excess of 100-fold oncentrations in excess of 100-fold greater than those needed to treat greater than those needed to treat the viralthe viral infection.infection.
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• The drug may be administered orally or The drug may be administered orally or by intravenous infusion. by intravenous infusion. TheThe bioavailability from oral administration bioavailability from oral administration is goodis good, the drug being distributed into , the drug being distributed into most bodymost body fluids and tissues. fluids and tissues.
• However, when used to treat AIDS it However, when used to treat AIDS it has given rise to gastrointestinalhas given rise to gastrointestinal disorders, skin rashes, insomnia, disorders, skin rashes, insomnia, anaemia, fever, headaches, depression anaemia, fever, headaches, depression and otherand other unwanted effects.unwanted effects.
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ResistanceResistance
•Resistance increases with time. Resistance increases with time. This is known to be due to This is known to be due to the the virusvirus developing mutations’developing mutations’ which result in changes in the which result in changes in the amino acid sequences in the amino acid sequences in the reversereverse transcriptase.transcriptase.
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DidanosineDidanosine
• Didanosine is used to treat some Didanosine is used to treat some AZT-resistant strains of HIV. It is also AZT-resistant strains of HIV. It is also used inused in combination with AZT to combination with AZT to treat HIV. Didanosine is administered treat HIV. Didanosine is administered orally in dosage forms thatorally in dosage forms that contain contain antacid buffers to prevent conversion antacid buffers to prevent conversion by the stomach acids to by the stomach acids to hypoxanthinehypoxanthine
prof. azaprof. aza
• However, in spite of the use of However, in spite of the use of buffers the bioavailability from oral buffers the bioavailability from oral administration isadministration is low. low.
• The drug can cause nausea, The drug can cause nausea, abdominal pain and peripheral abdominal pain and peripheral neuropathy, amongst otherneuropathy, amongst other symptoms. symptoms. Drug resistance occurs Drug resistance occurs after prolonged use.after prolonged use.
prof. azaprof. aza
prof. azaprof. aza
• Didanosine is converted by viral and Didanosine is converted by viral and cellular kinases to the monophosphate cellular kinases to the monophosphate and then toand then to the triphosphate. In this the triphosphate. In this form it inhibits reverse transcriptase form it inhibits reverse transcriptase and in addition itsand in addition its incorporation into incorporation into the DNA chain terminates the chain the DNA chain terminates the chain because the drug has no 30-hydroxybecause the drug has no 30-hydroxy group (Fig. 10.45).group (Fig. 10.45).
prof. azaprof. aza
Host cell penetration inhibitorsHost cell penetration inhibitors
• The principal drugs that act in this The principal drugs that act in this manner are amantadine and manner are amantadine and rimantadine (Fig. 10.46).rimantadine (Fig. 10.46).
• Both amantadine and rimantadine Both amantadine and rimantadine are also used to treat Parkinson’s are also used to treat Parkinson’s disease. However, theirdisease. However, their mode of mode of action in this disease is different from action in this disease is different from their action as antiviral agents.their action as antiviral agents.
prof. azaprof. aza
prof. azaprof. aza
Amantadine Amantadine hydrochloridehydrochloride
•Amantadine hydrochloride is Amantadine hydrochloride is effective against influenza Aeffective against influenza A virus virus but is not effective against the but is not effective against the influenza B virus. When used as a influenza B virus. When used as a prophylactic, it isprophylactic, it is believed to give believed to give up to 80 per cent protection up to 80 per cent protection against influenza A virus infectionsagainst influenza A virus infections
prof. azaprof. aza
prof. azaprof. aza
•The drugThe drug acts by blocking an ion acts by blocking an ion channel in the virus membrane channel in the virus membrane formed by the viral proteinM2. formed by the viral proteinM2. This isThis is believed to inhibit the believed to inhibit the disassembly of the core of the disassembly of the core of the virion and its penetration of the virion and its penetration of the host (seehost (see section 10.14.1).section 10.14.1).
prof. azaprof. aza
• Amantadine hydrochloride has a good Amantadine hydrochloride has a good bioavailability on oral administration, bioavailability on oral administration, beingbeing readily absorbed and distributed readily absorbed and distributed to most body fluids and tissues. to most body fluids and tissues.
• Its elimination time isIts elimination time is 12–18 hours. 12–18 hours. However, its use can result in However, its use can result in depression, dizziness, insomnia anddepression, dizziness, insomnia and gastrointestinal disturbances, amongst gastrointestinal disturbances, amongst other unwanted side effects.other unwanted side effects.
prof. azaprof. aza
Rimantadine Rimantadine hydrochloridehydrochloride
•Rimantadine hydrochloride is an Rimantadine hydrochloride is an analogue of amantadineanalogue of amantadine hydrochloride. It is more hydrochloride. It is more effective against influenza A effective against influenza A virus than amantadine. Its mode virus than amantadine. Its mode ofof action is probably similar to action is probably similar to that of amantadine.that of amantadine.
prof. azaprof. aza
•The drug is readily absorbed whenThe drug is readily absorbed when administered orally but undergoes administered orally but undergoes extensive first-pass metabolism. extensive first-pass metabolism. However, in spite ofHowever, in spite of this, its this, its elimination half-life is double that elimination half-life is double that of amantadine. Furthermore, CNS of amantadine. Furthermore, CNS side effects areside effects are significantly significantly reduced.reduced.
prof. azaprof. aza
Inhibitors of viral protein Inhibitors of viral protein synthesissynthesis
• The principal compounds that act as The principal compounds that act as inhibitors of protein synthesis are the inhibitors of protein synthesis are the interferons.interferons.
• These compounds are members of a These compounds are members of a naturally occurring family of naturally occurring family of glycoprotein hormonesglycoprotein hormones (RMM 20 (RMM 20 000–160 000), which are produced 000–160 000), which are produced by nearly all types of eukaryotic cell.by nearly all types of eukaryotic cell.
prof. azaprof. aza
• Three general classes of interferons Three general classes of interferons are known are known to occur naturally in to occur naturally in mammals, namelymammals, namely:: the the αα-interferons -interferons produced by leucocytes, produced by leucocytes, ββ-interferons -interferons produced by fibroblasts andproduced by fibroblasts and γ-γ-interferons interferons produced by T produced by T lymphocyteslymphocytes. At least twenty . At least twenty αα-, two -, two ββ- - and two and two γγ-interferons-interferons have been have been identifiedidentified
prof. azaprof. aza
• Interferons form part of the human Interferons form part of the human immune system. It is believed that the immune system. It is believed that the presence ofpresence of virions, bacteria and other virions, bacteria and other antigens in the body switches on the antigens in the body switches on the mRNA that controls themRNA that controls the production production and release of interferon. This release and release of interferon. This release stimulates other cells to produce andstimulates other cells to produce and
• release more interferon.release more interferon.
prof. azaprof. aza
• Interferons are thought to act by Interferons are thought to act by initiating the production in theinitiating the production in the cell of proteins that protect the cell of proteins that protect the cells from viral attack. The main cells from viral attack. The main action of these proteinsaction of these proteins takes takes the form of inhibiting the the form of inhibiting the synthesis of viral mRNA and viral synthesis of viral mRNA and viral protein synthesis. protein synthesis.
prof. azaprof. aza
•a- Interferons also enhance a- Interferons also enhance the activity of killer T cells the activity of killer T cells associated with the immune associated with the immune system. (see section 14.5.5).system. (see section 14.5.5).
prof. azaprof. aza
•The main action of these proteins The main action of these proteins takes the form of takes the form of inhibiting the inhibiting the synthesis of viral mRNA and viral synthesis of viral mRNA and viral protein synthesisprotein synthesis. .
•αα- Interferons also - Interferons also enhance the enhance the activity of killer T cells activity of killer T cells associated associated with the immune system.with the immune system.
prof. azaprof. aza
•A number of a-interferons have A number of a-interferons have been manufactured andbeen manufactured and proven proven to be reasonably effective to be reasonably effective against a number of viruses and against a number of viruses and cancers. cancers.
• Interferons areInterferons are usually given by usually given by intravenous, intramuscular or intravenous, intramuscular or subcutaneous injection.subcutaneous injection.
prof. azaprof. aza
• However, theirHowever, their administration can administration can cause adverse effects, such as cause adverse effects, such as headaches, fevers and bone marrowheadaches, fevers and bone marrow depression, that are dose related.depression, that are dose related.
• The formation and release of The formation and release of interferon by viral and other interferon by viral and other pathological stimulation haspathological stimulation has resulted resulted in a search for chemical inducers of in a search for chemical inducers of endogenous interferon.endogenous interferon.
prof. azaprof. aza
•Administration of aAdministration of a wide range of wide range of compounds has resulted in the compounds has resulted in the induction of interferon production. induction of interferon production. However,However, no clinically useful no clinically useful compounds have been found for compounds have been found for humans’ although tilorone is humans’ although tilorone is effectiveeffective in inducing interferon in inducing interferon in in mice.mice.
prof. azaprof. aza
prof. azaprof. aza
10.15. 10.15. Recombinant DNA (Genetic Recombinant DNA (Genetic Engineering)Engineering)
• The body requires a constant supply of The body requires a constant supply of certain peptides and proteins if it is to certain peptides and proteins if it is to remain remain healthhealth and function normally. and function normally. Many oMany off these pepti these peptiddes and proteins es and proteins are only produced in are only produced in aa small small quantities. quantities. They will They will bbe produced only e produced only if the correct genes are present in the if the correct genes are present in the cell. cell. CCononsequentlysequently, , if a gene is missing if a gene is missing or defective an essential protein willor defective an essential protein will
• not be produced, not be produced, which can lead to a which can lead to a diseased statediseased state..
•Consequently, if a gene is Consequently, if a gene is missing or defective an missing or defective an essential protein willessential protein will not be not be produced, produced, which can lead to a which can lead to a diseased state.diseased state.
prof. azaprof. aza
prof. azaprof. aza
•For example, cystic fibrosis is For example, cystic fibrosis is causedcaused by a defective gene. by a defective gene. This This faulty gene produces a defective faulty gene produces a defective membrane protein, membrane protein, cysticcystic fibrosis fibrosis transmembrane regulator transmembrane regulator (CFTR), which will not allow the (CFTR), which will not allow the free passage of chloridefree passage of chloride ions ions through the membranethrough the membrane
• The passage of chloride ions through The passage of chloride ions through a normal membrane intoa normal membrane into the lungs is the lungs is usually accompanied by a flow of usually accompanied by a flow of water molecules in the same water molecules in the same directiondirection. .
• InIn membranes that contain CFTR the membranes that contain CFTR the transport of water through the transport of water through the membrane into the lungsmembrane into the lungs is reduced.is reduced.
prof. azaprof. aza
•This viscous mucus clogs the This viscous mucus clogs the lungslungs and and makes breathing makes breathing difficultdifficult, a classic symptom of , a classic symptom of cystic fibrosis. It also provides cystic fibrosis. It also provides aa breeding ground for bacteria that breeding ground for bacteria that cause pneumonia cause pneumonia and other and other illnesses.illnesses.
prof. azaprof. aza
• Several thousand Several thousand hereditary diseases hereditary diseases found in humans are known to be found in humans are known to be caused by faultycaused by faulty genes. Recombinant genes. Recombinant DNA (rDNA) technology (genetic DNA (rDNA) technology (genetic engineering) offers a new way ofengineering) offers a new way of combating these hereditary diseases combating these hereditary diseases by either replacing the faulty genes or by either replacing the faulty genes or producing theproducing the missing peptides and missing peptides and proteinsproteins so that they can be given as a so that they can be given as a medicine (see section 10.15.2).medicine (see section 10.15.2).
prof. azaprof. aza
•The first step in any use of The first step in any use of recombinant DNA technology is to recombinant DNA technology is to isolate or copy theisolate or copy the required gene. required gene. There are three sources of the There are three sources of the genes required for cloning. The genes required for cloning. The two mosttwo most important are genomic important are genomic and copy or and copy or complementary DNA complementary DNA (cDNA) libraries.(cDNA) libraries.
prof. azaprof. aza
• In the first caseIn the first case the library the library consists of DNA fragments consists of DNA fragments obtained from a cell’s genome, obtained from a cell’s genome, whilst in the secondwhilst in the second case the case the library consists of DNA fragments library consists of DNA fragments synthesised by using the mRNA synthesised by using the mRNA for the proteinfor the protein of interestof interest..
prof. azaprof. aza
•The third is by the automated The third is by the automated synthesis of DNA, which is only synthesis of DNA, which is only feasible if thefeasible if the required base required base sequence is known. This may be sequence is known. This may be deduced from the amino acid deduced from the amino acid sequence ofsequence of the required protein the required protein if it is known.if it is known.
prof. azaprof. aza
•Once the gene has been obtained Once the gene has been obtained it is inserted into ait is inserted into a carrier (vector) carrier (vector) that can enter a host cell and be that can enter a host cell and be replicated, propagated and replicated, propagated and transcripted intotranscripted into mRNA by the mRNA by the cellular biochemistry of that cell. cellular biochemistry of that cell. This process is often referred to This process is often referred to as geneas gene cloning.cloning.
prof. azaprof. aza
• The mRNA produced by the cloned The mRNA produced by the cloned DNA is used by the cell ribosomes to DNA is used by the cell ribosomes to produce the protein encoded by the produce the protein encoded by the cloned DNA. In theory, cloned DNA. In theory, gene cloninggene cloning makes it possible to produce any makes it possible to produce any protein provided that it is possible to protein provided that it is possible to obtain a copy of theobtain a copy of the corresponding corresponding genegene. Products produced using . Products produced using recombinant DNA usually have recombinant DNA usually have recombinant,recombinant, r or rDNA in their r or rDNA in their names.names. prof. azaprof. aza
15.1. Gene cloning15.1. Gene cloning
•Bacteria are frequently used as host Bacteria are frequently used as host cells for gene cloning. This is cells for gene cloning. This is because they normally usebecause they normally use the the same genetic code as humans to same genetic code as humans to make peptides and proteins. make peptides and proteins. However, in bacteria theHowever, in bacteria the mechanism mechanism for peptide and protein formation is for peptide and protein formation is somewhat different.somewhat different.
prof. azaprof. aza
• It is not restricted toIt is not restricted to the the chromosomes but can also occur chromosomes but can also occur in extranuclear particles called in extranuclear particles called plasmids. Plasmids areplasmids. Plasmids are large large circular supercoiled DNA circular supercoiled DNA molecules whose structure molecules whose structure contains at least one gene and acontains at least one gene and a start site for replication.start site for replication.
prof. azaprof. aza
•However, the number of genes However, the number of genes found in a plasmid is fairly found in a plasmid is fairly limited,limited, although bacteria will although bacteria will contain a number of identical contain a number of identical copies of the same plasmid.copies of the same plasmid.
• It is possible to isolate the It is possible to isolate the plasmids of bacterial cells.plasmids of bacterial cells.
prof. azaprof. aza
•The isolated DNA molecules can beThe isolated DNA molecules can be
•broken open by cleaving the broken open by cleaving the phosphate bonds between specific phosphate bonds between specific pairs of bases by the actionpairs of bases by the action enzymes known as restriction enzymes known as restriction enzymes or endonucleasesenzymes or endonucleases
prof. azaprof. aza
•Each of these enzymes, of whichEach of these enzymes, of which over 500 are known, will only over 500 are known, will only cleave the bonds between specific cleave the bonds between specific nucleosides. For example,nucleosides. For example, EcoR I EcoR I cleaves the phosphate link between cleaves the phosphate link between guanosine and adenosine whilst guanosine and adenosine whilst Xho I cuts theXho I cuts the chain between chain between cytidine and thymine nucleosides.cytidine and thymine nucleosides.
prof. azaprof. aza
•Cutting the strand can result in Cutting the strand can result in either blunteither blunt ends, where the ends, where the endonuclease cuts across both endonuclease cuts across both chains of the DNA at the same chains of the DNA at the same points, orpoints, or cohesive ends (sticky cohesive ends (sticky ends), where the cut is ends), where the cut is staggered from one chain to the staggered from one chain to the otherother (Fig. 10.47).(Fig. 10.47).
prof. azaprof. aza
•The new non-cyclic structure of The new non-cyclic structure of the plasmid is known as the plasmid is known as linearised DNA inlinearised DNA in order to order to distinguish it from the new insert distinguish it from the new insert or foreign DNA. or foreign DNA.
prof. azaprof. aza
• This foreign DNA must contain the This foreign DNA must contain the required gene, a second gene system required gene, a second gene system that confers resistance to a specific that confers resistance to a specific antibiotic and any other necessary antibiotic and any other necessary information. It should be remembered information. It should be remembered that a eukaryotic gene is made upthat a eukaryotic gene is made up of of exons separated by introns, which are exons separated by introns, which are sequences that have no apparent use.sequences that have no apparent use.
prof. azaprof. aza
• Figure 10.47 (a) Blunt and (b) cohesive Figure 10.47 (a) Blunt and (b) cohesive cuts with compatible adhesive cutscuts with compatible adhesive cuts
prof. azaprof. aza
• Mixing the foreign DNA and the Mixing the foreign DNA and the linearised DNA in a suitable medium linearised DNA in a suitable medium results in theresults in the formation of extended formation of extended plasmid loops when their ends come plasmid loops when their ends come into contact (Fig. 10.48). Thisinto contact (Fig. 10.48). This contact contact is converted into a permanent bond is converted into a permanent bond by the catalytic action of an enzyme by the catalytic action of an enzyme calledcalled DNA ligase.DNA ligase.
prof. azaprof. aza
• Figure Figure 10.48. A 10.48. A represenrepresentation of tation of the main the main steps in steps in the the insertion insertion of a gene of a gene into a into a plasmidplasmid
prof. azaprof. aza
•ThisThis contact is converted into a contact is converted into a permanent bond by the catalytic permanent bond by the catalytic action of an enzyme calledaction of an enzyme called DNA DNA ligase. When the chains are ligase. When the chains are cohesive the exposed single cohesive the exposed single chains of new DNA mustchains of new DNA must contain contain a complementary base sequence a complementary base sequence to the exposed ends of the to the exposed ends of the linearised DNAlinearised DNA..
prof. azaprof. aza
• TheThe hydrogen bonding between hydrogen bonding between these complementary base pairs these complementary base pairs tends to bind the chainstends to bind the chains together together prior to the action of the DNA ligase, prior to the action of the DNA ligase, hence the name ‘‘sticky ends’’. The hence the name ‘‘sticky ends’’. The newnew DNA of the modified plasmid is DNA of the modified plasmid is known as recombinant DNA (rDNA).known as recombinant DNA (rDNA).
prof. azaprof. aza
• However, the randomHowever, the random nature of the nature of the techniques used to form the modified techniques used to form the modified plasmids means that some of theplasmids means that some of the linearised DNA reforms the plasmid linearised DNA reforms the plasmid without incorporating the foreign without incorporating the foreign DNA, that is, aDNA, that is, a mixture of both types mixture of both types of plasmid is formed.of plasmid is formed.
prof. azaprof. aza
•The modified plasmids are The modified plasmids are separated from theseparated from the unmodified unmodified plasmids when they are plasmids when they are reinserted into a bacterial cell.reinserted into a bacterial cell.
prof. azaprof. aza
• The new plasmids are reinserted into The new plasmids are reinserted into the bacteria by a process known as the bacteria by a process known as transformation.transformation.
• Bacteria are mixed with the new Bacteria are mixed with the new plasmids in a medium containing plasmids in a medium containing calcium chloride. Thiscalcium chloride. This medium makes medium makes the bacterial membrane permeable the bacterial membrane permeable to the plasmidto the plasmid..
prof. azaprof. aza
•However, not all bacteriaHowever, not all bacteria will will take up the modified plasmids. take up the modified plasmids. Such bacteria can easily be Such bacteria can easily be destroyed by specific antibioticdestroyed by specific antibiotic action since they do not contain action since they do not contain plasmids with the appropriate plasmids with the appropriate protecting gene.protecting gene.
prof. azaprof. aza
•This makesThis makes isolation of the isolation of the bacteria with the modified bacteria with the modified plasmids relatively simple. plasmids relatively simple.
•These modified bacteriaThese modified bacteria are are allowed to replicate and, in doing allowed to replicate and, in doing so, produce many copies of the so, produce many copies of the modified plasmid.modified plasmid.
prof. azaprof. aza
•UnderUnder favourable conditions one favourable conditions one modified bacterial cell can produce modified bacterial cell can produce over 200 copies of the newover 200 copies of the new plasmid. The gene in these plasmid. The gene in these modified plasmids will use the modified plasmids will use the bacteria’s internal machinery tobacteria’s internal machinery to automatically produce the automatically produce the appropriate peptide or protein.appropriate peptide or protein.
prof. azaprof. aza
•Since many bacteria replicate at Since many bacteria replicate at aa very rapid rate this technique very rapid rate this technique offers a relatively quick way of offers a relatively quick way of producing large quantities ofproducing large quantities of essential naturally occurring essential naturally occurring compounds that cannot be compounds that cannot be produced by other means.produced by other means.
prof. azaprof. aza
•Plasmids are not the only vectors Plasmids are not the only vectors that can be used to transport that can be used to transport DNA into a bacterial hostDNA into a bacterial host cell. cell.
•Foreign DNA can also be inserted Foreign DNA can also be inserted into bacteriophages and cosmids into bacteriophages and cosmids by similarby similar techniques.techniques.
prof. azaprof. aza
• Bacteriophages (phage) are viruses Bacteriophages (phage) are viruses that specifically infect bacteria whilst that specifically infect bacteria whilst aa cosmid is a hybrid between a cosmid is a hybrid between a phage and a plasmid that has been phage and a plasmid that has been especially synthesised forespecially synthesised for use in use in gene cloning. Plasmids can be used gene cloning. Plasmids can be used to insert fragments containing up to to insert fragments containing up to 10 kilobasepairs10 kilobasepairs (kbp), phages up to (kbp), phages up to 20 kbp and cosmids 50 or more kbp.20 kbp and cosmids 50 or more kbp.
prof. azaprof. aza
• It is not always necessary to use a It is not always necessary to use a vector to place the recombinant vector to place the recombinant DNA in a cell. If theDNA in a cell. If the cell is large cell is large enough, the recombinant DNA enough, the recombinant DNA may be placed in the cell by using may be placed in the cell by using aa micropipette whose overall tip micropipette whose overall tip diameter is less than 1 mm.diameter is less than 1 mm.
prof. azaprof. aza
•Only a small amount of theOnly a small amount of the recombinant DNA inserted in this recombinant DNA inserted in this fashion is taken up by the cell’s fashion is taken up by the cell’s chromosomes. However,chromosomes. However, this this small fraction will increase to a small fraction will increase to a significant level as the cell significant level as the cell replicates (Fig. 10.48).replicates (Fig. 10.48).
prof. azaprof. aza
• Host cells for all methods of cloning are Host cells for all methods of cloning are usually either bacterial or mammalian usually either bacterial or mammalian in origin. For example, bacterial cells in origin. For example, bacterial cells often used are E. coli and eukaryotic often used are E. coli and eukaryotic yeast while mammalian cell lines yeast while mammalian cell lines include Chinese hamster ovary (CHO), include Chinese hamster ovary (CHO), baby hamster kidney (BHK) and African baby hamster kidney (BHK) and African green monkey kidney (VERO).green monkey kidney (VERO).
prof. azaprof. aza
• In all cases small-scale cultures In all cases small-scale cultures of the host cell plus vectorof the host cell plus vector are are grown to find the culture grown to find the culture containing the host with the containing the host with the required gene that gives the bestrequired gene that gives the best yield of the desired protein.yield of the desired protein.
prof. azaprof. aza
•Once this culture has been Once this culture has been determined the process is scaled determined the process is scaled up via a suitable pilot plant to up via a suitable pilot plant to production level (see section production level (see section 16.6). The mammalian cell line 16.6). The mammalian cell line cultures normally give poorer cultures normally give poorer yields of the desired protein.yields of the desired protein.
prof. azaprof. aza
prof. azaprof. aza
15.2.2 Manufacture of Pharmaceuticals15.2.2 Manufacture of Pharmaceuticals
• The bodyThe body produces peptides and produces peptides and proteproteinsins, often in extremely small , often in extremely small quantitiesquantities, which , which are essential for its are essential for its well being. The absence of the well being. The absence of the necessary’ genes means that the body necessary’ genes means that the body does not produce these essential does not produce these essential compounds, resulting in a deficiency compounds, resulting in a deficiency disease that is usually’ fatal. disease that is usually’ fatal.
• Treatment by supplying the patient Treatment by supplying the patient with sufficient amounts of twith sufficient amounts of thehe missing missing compounds is normally successful. compounds is normally successful.
•However, extraction from other However, extraction from other natural sources is usually’ natural sources is usually’ difficult and yields are often low. difficult and yields are often low. For exampleFor example,, it takes half a it takes half a million sheep brains to produce million sheep brains to produce 5mg of somatostatin5mg of somatostatin a growth a growth horhormmone that inhibits secretioone that inhibits secretionn of the pituitary growof the pituitary growthth hormone hormone. .
prof. azaprof. aza
•FurtFurthhermore, unless termore, unless thehe source source of the required product is of the required product is donated blood there is a limit to donated blood there is a limit to the number of cadavers available the number of cadavers available for the extraction of compoufor the extraction of compounnds ds suitable for use in husuitable for use in hummans. ans.
prof. azaprof. aza
• Moreover, there is also the danger Moreover, there is also the danger that compounds obtained from that compounds obtained from human sources human sources maymay bbe contaminated e contaminated by’ viruses such as HIVby’ viruses such as HIV,, hepatitis hepatitis, , Creutzfeld–Jakob disease (mad cowCreutzfeld–Jakob disease (mad cow disease)disease) and others tand others thhat are difficult at are difficult to detect. Animal sources have been to detect. Animal sources have been used but only a few human protein used but only a few human protein deficiency disorders deficiency disorders cancan bbe treated e treated withwith animal proteins. animal proteins.
prof. azaprof. aza
prof. azaprof. aza
• Gene cloGene clonniinng is used to obtain human g is used to obtain human recombirecombinnant proteins. Howeverant proteins. However,, some some proteins will also need post—proteins will also need post—translational motranslational modification dification such as such as glycosylation and/or tglycosylation and/or the modificationhe modification of aof ammino acid sequences. These ino acid sequences. These modifications may require formodifications may require formming ing different section, of the peptide chain different section, of the peptide chain in tin thhe culture e culture mmedium and cedium and chemihemically’ cally’ combining these sectiocombining these sections in vitrons in vitro. .
• The genes required for these The genes required for these processes are synthesised usiprocesses are synthesised usinng the g the required prequired peeptide aptide ass a blueprint. For a blueprint. For example, human recombinant example, human recombinant insulinsulineine may he produced in t may he produced in thihis s manner (Figure 10.12). The genes for manner (Figure 10.12). The genes for the A and B chaithe A and B chainns of insulin were s of insulin were syntsynthhesised separately. esised separately.
prof. azaprof. aza
• TThhey were cloned separately, using ey were cloned separately, using suitable plasmids. into two different suitable plasmids. into two different bacterial strains. One of these strains bacterial strains. One of these strains is is usedused to produce t to produce thehe A chain A chain whilst whilst the the othersothers is used to produce the B is used to produce the B strain. strain. TheThe chains are isolated and chains are isolated and attached to each other byattached to each other by in vitro in vitro didisulphide bond formation. sulphide bond formation.
prof. azaprof. aza
•This last step is inefficient and This last step is inefficient and human recombinanthuman recombinant insulin is insulin is now made by forming now made by forming recombinant proinsulin by gene recombinant proinsulin by gene cloning. The proinsulin iscloning. The proinsulin is converted to recombinant insulin converted to recombinant insulin by proteolytic cleavageby proteolytic cleavage
prof. azaprof. aza
prof. azaprof. aza
Figure 10.42. An outline of the synthesis of recombinant human insulin.